p subalpina strain uamh 11012  (Roche)


Bioz Verified Symbol Roche is a verified supplier
Bioz Manufacturer Symbol Roche manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 85

    Structured Review

    Roche p subalpina strain uamh 11012
    Map of the mt genome of Phialocephala <t>subalpina.</t> Map displaying the circular mt genome of P. subalpina strain UAMH 11012. All open reading frames, tRNA genes and the large ribosomal RNA are transcribed clockwise.
    P Subalpina Strain Uamh 11012, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 1516 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p subalpina strain uamh 11012/product/Roche
    Average 85 stars, based on 1516 article reviews
    Price from $9.99 to $1999.99
    p subalpina strain uamh 11012 - by Bioz Stars, 2020-09
    85/100 stars

    Images

    1) Product Images from "Mitochondrial genome evolution in species belonging to the Phialocephala fortinii s.l. - Acephala applanata species complex"

    Article Title: Mitochondrial genome evolution in species belonging to the Phialocephala fortinii s.l. - Acephala applanata species complex

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-13-166

    Map of the mt genome of Phialocephala subalpina. Map displaying the circular mt genome of P. subalpina strain UAMH 11012. All open reading frames, tRNA genes and the large ribosomal RNA are transcribed clockwise.
    Figure Legend Snippet: Map of the mt genome of Phialocephala subalpina. Map displaying the circular mt genome of P. subalpina strain UAMH 11012. All open reading frames, tRNA genes and the large ribosomal RNA are transcribed clockwise.

    Techniques Used:

    2) Product Images from "Mitochondrial genome evolution in species belonging to the Phialocephala fortinii s.l. - Acephala applanata species complex"

    Article Title: Mitochondrial genome evolution in species belonging to the Phialocephala fortinii s.l. - Acephala applanata species complex

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-13-166

    Map of the mt genome of Phialocephala subalpina. Map displaying the circular mt genome of P. subalpina strain UAMH 11012. All open reading frames, tRNA genes and the large ribosomal RNA are transcribed clockwise.
    Figure Legend Snippet: Map of the mt genome of Phialocephala subalpina. Map displaying the circular mt genome of P. subalpina strain UAMH 11012. All open reading frames, tRNA genes and the large ribosomal RNA are transcribed clockwise.

    Techniques Used:

    3) Product Images from "Polyclonality of BRAF Mutations in Acquired Melanocytic Nevi"

    Article Title: Polyclonality of BRAF Mutations in Acquired Melanocytic Nevi

    Journal: JNCI Journal of the National Cancer Institute

    doi: 10.1093/jnci/djp309

    Polyclonality of v-raf murine sarcoma viral oncogene homolog B1 ( BRAF ) mutations in acquired melanocytic nevi. A ) Selection of single nevus cells after immunomagnetic separation. Single nevus cells ( purple dots with arrows ) were captured by high molecular weight-melanoma-associated antigen–specific monoclonal antibodies bound to immunomagnetic beads ( pink dots ). The cells ( encircled ) were procured by laser-capture microdissection ( top ; bar = 20 μm). Polymerase chain reaction (PCR) amplification and subsequent sequencing of single nevus cells showed wild-type BRAF and BRAF V600E mutations ( bottom ). B ) Laser-capture microdissection of frozen tissue section of acquired melanocytic nevi followed by direct sequencing of BRAF exon 15 ( top ; bar = 20 μm). Sequencing revealed two of the contiguous single nevus cells to have the BRAF V600E mutation and one to have a compound heterozygous BRAF V600E (T1799A) and BRAF V600G (T1799G) mutation, showing a heterogeneous pattern of BRAF mutations in proximal cells on a single-cell level ( bottom ). C ) Subcloning and subsequent sequencing of BRAF exon 15 and the single nucleotide polymorphism (SNP) rs7801086. This SNP maps approximately 2 kb telomeric from BRAF exon 15. Four nevi (numbers 3, 6, 11, and 14) were excised from patients who were heterozygous for this SNP. DNA was extracted from hundreds of nevus cells isolated either by using immunomagnetic beads (numbers 3 and 6) or laser-capture microdissection of frozen tissue sections (numbers 11 and 14). A 2859-bp fragment containing both BRAF exon 15 and the SNP rs7801086 was amplified by long-range PCR. Subcloning was carried out using this fragment as an insert. Sixteen to 30 colonies were randomly picked from each patient sample and analyzed for the sequence of both BRAF exon 15 and rs7801086. In all four patient samples, colonies with BRAF V600E as well as wild-type BRAF were accompanied by different SNP alleles, some harboring the G allele and others harboring the T allele. In sample number 14, one colony (*) showed a tandem BRAF V600E/K601E (T1799→A and A1802→G) mutation.
    Figure Legend Snippet: Polyclonality of v-raf murine sarcoma viral oncogene homolog B1 ( BRAF ) mutations in acquired melanocytic nevi. A ) Selection of single nevus cells after immunomagnetic separation. Single nevus cells ( purple dots with arrows ) were captured by high molecular weight-melanoma-associated antigen–specific monoclonal antibodies bound to immunomagnetic beads ( pink dots ). The cells ( encircled ) were procured by laser-capture microdissection ( top ; bar = 20 μm). Polymerase chain reaction (PCR) amplification and subsequent sequencing of single nevus cells showed wild-type BRAF and BRAF V600E mutations ( bottom ). B ) Laser-capture microdissection of frozen tissue section of acquired melanocytic nevi followed by direct sequencing of BRAF exon 15 ( top ; bar = 20 μm). Sequencing revealed two of the contiguous single nevus cells to have the BRAF V600E mutation and one to have a compound heterozygous BRAF V600E (T1799A) and BRAF V600G (T1799G) mutation, showing a heterogeneous pattern of BRAF mutations in proximal cells on a single-cell level ( bottom ). C ) Subcloning and subsequent sequencing of BRAF exon 15 and the single nucleotide polymorphism (SNP) rs7801086. This SNP maps approximately 2 kb telomeric from BRAF exon 15. Four nevi (numbers 3, 6, 11, and 14) were excised from patients who were heterozygous for this SNP. DNA was extracted from hundreds of nevus cells isolated either by using immunomagnetic beads (numbers 3 and 6) or laser-capture microdissection of frozen tissue sections (numbers 11 and 14). A 2859-bp fragment containing both BRAF exon 15 and the SNP rs7801086 was amplified by long-range PCR. Subcloning was carried out using this fragment as an insert. Sixteen to 30 colonies were randomly picked from each patient sample and analyzed for the sequence of both BRAF exon 15 and rs7801086. In all four patient samples, colonies with BRAF V600E as well as wild-type BRAF were accompanied by different SNP alleles, some harboring the G allele and others harboring the T allele. In sample number 14, one colony (*) showed a tandem BRAF V600E/K601E (T1799→A and A1802→G) mutation.

    Techniques Used: Selection, Immunomagnetic Separation, Molecular Weight, Laser Capture Microdissection, Polymerase Chain Reaction, Amplification, Sequencing, Mutagenesis, Subcloning, Isolation

    4) Product Images from "Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells"

    Article Title: Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20131436

    Cellular senescence in Usp3 Δ/Δ HSC compartment and BM. (A) Cytospins of BM cells from Usp3 Δ/Δ and WT mice were assayed for SA-β-galactosidase activity. The percentage of SA- β -Gal–positive cells was quantified by counting 100 cells on three separate fields ( n = 4 mice per genotype). Bar, 20 µm. (B and C) Cytospin preparations of sorted LSKs from BM of Usp3 Δ/Δ or WT mice were immunostained for HP1γ (B) and H3K9Me3 (C). Focal nuclear staining is visible in insets. Signal intensity per nucleus was quantified by ImageJ. n = 3 per genotype. A minimum of 1,000 nuclei/sample was evaluated. Data are mean ± SEM of one of two representative experiments. Bars: 75 µm; (inset) 10 µm. (D) Immunostaining for HP1γ and H3K9Me3 on BM sections from Usp3 Δ/Δ and WT mice. The percentage of positive cells was quantified in 3 fields on a minimum of 1,500 cells/field per sample. n = 7 per genotype. Bar, 20 µm. (E) Quantification of apoptotic (Annexin V positive and Propidium Iodide [PI] negative) freshly isolated hematopoietic subpopulations (mean ± SD) from WT or Usp3 Δ/Δ. n = 3 per genotype. One of two representative experiments is shown. (F) Representative images of WT and Usp3 Δ/Δ BM sections stained for apoptosis-indicating cleavage (cl.) of caspase 3. n = 6 mice per genotype. Bar, 500 µm. (G) Sorted LT-HSCs were plated after 8 d (first plating) or 11 d (second plating) in culture and monitored for growth. Kinetic measures the number of cells, recorded over time and plotted as phase contrast object confluence. n = 4 wells per data point. Mean ± SD of one of two representative experiments is shown. Representative images at time 0 and 96 h after plating are shown. Bar, 300 µm. (H) Immunostaining of in vitro expanded LT-HSCs for H3K9Me3. Signal quantification by ImageJ from two independent experiments is shown (mean ± SEM). n = 150 per genotype. Bar, 10 µm. (I) LT-HSCs cultures were assayed for SA-β-galactosidase activity after 3, 8, or 11 d (dd) in culture. A minimum of 350 (3dd), 2300 (8 dd), or 550 (11 dd) cells counted in 10 separate fields were evaluated. Bar, 20 µm. (J) LT-HSCs cultures were assayed for SA-β-galactosidase activity upon Tat-cMyc protein transduction. A minimum of 1,000 cells per genotype was evaluated in two replicate experiments. Bar, 20 µm. Mice were 32 wk old (A–D) or 40–44 wk old (E and F). G–J: LT-HSCs for in vitro expansion were isolated from 40–44-wk-old mice. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001; ns, not significant.
    Figure Legend Snippet: Cellular senescence in Usp3 Δ/Δ HSC compartment and BM. (A) Cytospins of BM cells from Usp3 Δ/Δ and WT mice were assayed for SA-β-galactosidase activity. The percentage of SA- β -Gal–positive cells was quantified by counting 100 cells on three separate fields ( n = 4 mice per genotype). Bar, 20 µm. (B and C) Cytospin preparations of sorted LSKs from BM of Usp3 Δ/Δ or WT mice were immunostained for HP1γ (B) and H3K9Me3 (C). Focal nuclear staining is visible in insets. Signal intensity per nucleus was quantified by ImageJ. n = 3 per genotype. A minimum of 1,000 nuclei/sample was evaluated. Data are mean ± SEM of one of two representative experiments. Bars: 75 µm; (inset) 10 µm. (D) Immunostaining for HP1γ and H3K9Me3 on BM sections from Usp3 Δ/Δ and WT mice. The percentage of positive cells was quantified in 3 fields on a minimum of 1,500 cells/field per sample. n = 7 per genotype. Bar, 20 µm. (E) Quantification of apoptotic (Annexin V positive and Propidium Iodide [PI] negative) freshly isolated hematopoietic subpopulations (mean ± SD) from WT or Usp3 Δ/Δ. n = 3 per genotype. One of two representative experiments is shown. (F) Representative images of WT and Usp3 Δ/Δ BM sections stained for apoptosis-indicating cleavage (cl.) of caspase 3. n = 6 mice per genotype. Bar, 500 µm. (G) Sorted LT-HSCs were plated after 8 d (first plating) or 11 d (second plating) in culture and monitored for growth. Kinetic measures the number of cells, recorded over time and plotted as phase contrast object confluence. n = 4 wells per data point. Mean ± SD of one of two representative experiments is shown. Representative images at time 0 and 96 h after plating are shown. Bar, 300 µm. (H) Immunostaining of in vitro expanded LT-HSCs for H3K9Me3. Signal quantification by ImageJ from two independent experiments is shown (mean ± SEM). n = 150 per genotype. Bar, 10 µm. (I) LT-HSCs cultures were assayed for SA-β-galactosidase activity after 3, 8, or 11 d (dd) in culture. A minimum of 350 (3dd), 2300 (8 dd), or 550 (11 dd) cells counted in 10 separate fields were evaluated. Bar, 20 µm. (J) LT-HSCs cultures were assayed for SA-β-galactosidase activity upon Tat-cMyc protein transduction. A minimum of 1,000 cells per genotype was evaluated in two replicate experiments. Bar, 20 µm. Mice were 32 wk old (A–D) or 40–44 wk old (E and F). G–J: LT-HSCs for in vitro expansion were isolated from 40–44-wk-old mice. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001; ns, not significant.

    Techniques Used: Mouse Assay, Activity Assay, Staining, Immunostaining, Isolation, In Vitro, Transduction

    USP3 protects HSCs from genotoxic stress in vivo and in vitro. (A–C) Age-matched (8 wk old) WT ( n = 12) and Usp3 Δ/Δ ( n = 13) mice were exposed to 7 Gy TBI and monitored for 28 d. (A) Kaplan Meier survival curve. P-value was determined by Log-rank test. (B) WBC counts of unirradiated or irradiated mice (WT, 28 d after TBI) and Usp3 Δ/Δ (at time of sacrifice due to illness after TBI). Results are mean ± SD from two independent experiments. (C) Representative images of hematoxylin-eosin (H E)–stained tissue sections of WT (28 d after TBI) and of Usp3 Δ/Δ (at the time of sacrifice due to illness after 7 Gy TBI) mice. Bars: (BM) 100 µm; (spleen) 50 µm; (small intestine) 50 µm; (heart) 500 µm; (inset) 50 µm. (D) CFU-C from BM cells of 8-wk-old WT or Usp3 Δ/Δ mice. Mice ( n = 3 per genotype) were left untreated or subjected to TBI (5Gy). 7 d after IR, BM cells were isolated and plated on methylcellulose with cytokines. Results are means ± SD. (E and F) Absolute numbers (2 femurs and 2 hips bones) of Lin − , LSKs, and HSCs in 44-wk-old mice that were left untreated (UN) or exposed to 5 Gy TBI (5Gy) and sacrificed after 24 h (E). UN, n = 11 per genotype; IR, WT, n = 3; Usp3 Δ/Δ, n = 4. (F) Increased cell death in Usp3 Δ/Δ LSKs, as determined by Annexin V staining in mice as in E. UN, n = 3 per genotype; IR, WT, n = 3; Usp3 Δ/Δ, n = 4. Results are means ± SEM. (G and H) Immunofluorescence of γH2AX and 53BP1 in WT or Usp3 Δ/Δ LT-HSCs in culture for 8–10 d. IRIFs were scored in LT-HSCs after mock treatment or at 30 min and 24 h after 2Gy of IR. The percentage of cells containing > 5 IRIFs are plotted ± SD. Representative images for 53BP1 staining are shown (H). A minimum of 50 cells/sample/experiment over two (γH2AX) or three (53BP1) independent experiments was evaluated. Bar, 10 µm. (I) Percentage of micronuclei in WT or Usp3 Δ/Δ LT-HSCs in culture for 8–10 d. Cells were irradiated with 2Gy and micronuclei scored at 24 h after IR. Results are means of three independent experiments ± SD on a minimum of 70 cells/genotype. Bar, 5 µm. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ P ≤ 0.001; ns, not significant.
    Figure Legend Snippet: USP3 protects HSCs from genotoxic stress in vivo and in vitro. (A–C) Age-matched (8 wk old) WT ( n = 12) and Usp3 Δ/Δ ( n = 13) mice were exposed to 7 Gy TBI and monitored for 28 d. (A) Kaplan Meier survival curve. P-value was determined by Log-rank test. (B) WBC counts of unirradiated or irradiated mice (WT, 28 d after TBI) and Usp3 Δ/Δ (at time of sacrifice due to illness after TBI). Results are mean ± SD from two independent experiments. (C) Representative images of hematoxylin-eosin (H E)–stained tissue sections of WT (28 d after TBI) and of Usp3 Δ/Δ (at the time of sacrifice due to illness after 7 Gy TBI) mice. Bars: (BM) 100 µm; (spleen) 50 µm; (small intestine) 50 µm; (heart) 500 µm; (inset) 50 µm. (D) CFU-C from BM cells of 8-wk-old WT or Usp3 Δ/Δ mice. Mice ( n = 3 per genotype) were left untreated or subjected to TBI (5Gy). 7 d after IR, BM cells were isolated and plated on methylcellulose with cytokines. Results are means ± SD. (E and F) Absolute numbers (2 femurs and 2 hips bones) of Lin − , LSKs, and HSCs in 44-wk-old mice that were left untreated (UN) or exposed to 5 Gy TBI (5Gy) and sacrificed after 24 h (E). UN, n = 11 per genotype; IR, WT, n = 3; Usp3 Δ/Δ, n = 4. (F) Increased cell death in Usp3 Δ/Δ LSKs, as determined by Annexin V staining in mice as in E. UN, n = 3 per genotype; IR, WT, n = 3; Usp3 Δ/Δ, n = 4. Results are means ± SEM. (G and H) Immunofluorescence of γH2AX and 53BP1 in WT or Usp3 Δ/Δ LT-HSCs in culture for 8–10 d. IRIFs were scored in LT-HSCs after mock treatment or at 30 min and 24 h after 2Gy of IR. The percentage of cells containing > 5 IRIFs are plotted ± SD. Representative images for 53BP1 staining are shown (H). A minimum of 50 cells/sample/experiment over two (γH2AX) or three (53BP1) independent experiments was evaluated. Bar, 10 µm. (I) Percentage of micronuclei in WT or Usp3 Δ/Δ LT-HSCs in culture for 8–10 d. Cells were irradiated with 2Gy and micronuclei scored at 24 h after IR. Results are means of three independent experiments ± SD on a minimum of 70 cells/genotype. Bar, 5 µm. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ P ≤ 0.001; ns, not significant.

    Techniques Used: In Vivo, In Vitro, Mouse Assay, Irradiation, Staining, Isolation, Immunofluorescence

    USP3 deletion leads to a genome-wide increase in mono-ubiquitinated H2A (uH2A) and H2B (uH2B) in mouse cells and tissues. (A) Immunostaining of WT and Usp3 Δ/Δ MEFs with anti-Ub (FK2) antibody (red) and DAPI (blue). The FK2 signal intensity per nucleus was quantified by ImageJ. A minimum of 1,000 cells/sample was analyzed. Data are means ± SEM of two independent MEF lines per genotype. Bars: 500 µm; (inset) 10 µm. (B) WT or Usp3 Δ/Δ MEFs were infected with control retrovirus (empty vector, ev) or with retrovirus expressing WT USP3 (WT-USP3) and immunostained with FK2. Representative images and FK2 signal quantification as in A. Right panel: immunoblot of MEFs WCE for USP3 and CDK4 (*, nonspecific protein band). Data are means ± SEM of two independent experiments with a minimum of 800 cells/genotype. Bar, 500 µm. (C) Immunoblot of core histone fraction from WT and Usp3 Δ/Δ MEFs. Quantification by ImageJ of the uH2A and uH2B signal normalized, respectively, to H2A or H2B, averaged from four (uH2A) or three (uH2B) independent MEF lines per genotype is shown. Data are means ± SD. (D) FK2 staining on freshly isolated BM cells from WT and Usp3 Δ/Δ mice ( n = 3 per genotype). Signal intensity was quantified as in A. WT, n = 555; Usp3 Δ/Δ, n = 938. Data are means ± SEM. (E) Immunoblot of core histones fraction from liver and spleen of WT and Usp3 Δ/Δ. uH2A and uH2B were quantified as in C. uH2A, n = 3 mice; uH2B, n = 2 mice per genotype. Data are means ± SD. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
    Figure Legend Snippet: USP3 deletion leads to a genome-wide increase in mono-ubiquitinated H2A (uH2A) and H2B (uH2B) in mouse cells and tissues. (A) Immunostaining of WT and Usp3 Δ/Δ MEFs with anti-Ub (FK2) antibody (red) and DAPI (blue). The FK2 signal intensity per nucleus was quantified by ImageJ. A minimum of 1,000 cells/sample was analyzed. Data are means ± SEM of two independent MEF lines per genotype. Bars: 500 µm; (inset) 10 µm. (B) WT or Usp3 Δ/Δ MEFs were infected with control retrovirus (empty vector, ev) or with retrovirus expressing WT USP3 (WT-USP3) and immunostained with FK2. Representative images and FK2 signal quantification as in A. Right panel: immunoblot of MEFs WCE for USP3 and CDK4 (*, nonspecific protein band). Data are means ± SEM of two independent experiments with a minimum of 800 cells/genotype. Bar, 500 µm. (C) Immunoblot of core histone fraction from WT and Usp3 Δ/Δ MEFs. Quantification by ImageJ of the uH2A and uH2B signal normalized, respectively, to H2A or H2B, averaged from four (uH2A) or three (uH2B) independent MEF lines per genotype is shown. Data are means ± SD. (D) FK2 staining on freshly isolated BM cells from WT and Usp3 Δ/Δ mice ( n = 3 per genotype). Signal intensity was quantified as in A. WT, n = 555; Usp3 Δ/Δ, n = 938. Data are means ± SEM. (E) Immunoblot of core histones fraction from liver and spleen of WT and Usp3 Δ/Δ. uH2A and uH2B were quantified as in C. uH2A, n = 3 mice; uH2B, n = 2 mice per genotype. Data are means ± SD. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

    Techniques Used: Genome Wide, Immunostaining, Infection, Plasmid Preparation, Expressing, Staining, Isolation, Mouse Assay

    USP3-deficient HSCs accumulate spontaneous DNA damage. (A–C) γH2AX immunostaining on sorted Lin − Sca1 + c-Kit + CD150 + flk2/CD135 − CD34 − (LT-HSC) or CD34 + (ST-HSC) from 44-wk-old (A and B) or 17-wk-old (C) mice. Representative images (A) of LT-HSCs and ST-HSCs from 44-wk-old mice and quantification of the number of γH2AX foci/cell in HSCs from 44-wk-old (B) or 17-wk-old mice (minimum of 200 cells per genotype; C). Results are from two independent experiments. n = 3 mice/genotype/experiment. Bar, 5 µm. (D–F) Alkaline comet assay on sorted Usp3 Δ/Δ LSKs (D and E) or total BM cells (F). Representative LSKs images (D) and the Average Tail Moment calculated by Comet Score on LSKs (E) or BM cells (F) are shown. A minimum of 150 comets was evaluated per sample. n = 3 per genotype, 44 wk old. Bar, 50 µm. (G–J) Sorted LT-HSCs from BM of 40–44 wk old mice were grown in liquid cultures and analyzed for DNA damage. (G) Immunostaining of γH2AX and 53BP1 on LT-HSCs after 8–11 d in culture. The percentage of cells containing > 5 γH2AX and 53BP1 foci was evaluated in three independent experiments. n > 50 cells/genotype/experiment. Arrows: γH2AX-53BP1 colocalizing foci. Bar, 5 µm. (H) Immunostaining of FK2 LT-HSCs after 8–11 d in culture. Representative images and quantification by Image J of FK2 signal intensity from three independent experiments. Nuclei are outlined. n > 100 cells/genotype/experiment. Bar, 10 µm. (I) Co-immunostaining of FK2 and 53BP1 on LT-HSCs after 8–11 d in culture. The number of co-foci (arrows) per cell was quantified in 2 independent experiments, on a total of n = 145 (WT) or 80 ( Usp3 Δ/Δ) cells scored. Bar, 10 µm. (J) Percentage of micronuclei in LT-HSCs cultures after 8 or 15 d in culture. A minimum of 70 cells/sample was scored in three independent experiments. Bars, 10 µm. In all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Results are mean ± SEM (B, E, F, H, and I) or mean ± SD (G and J).
    Figure Legend Snippet: USP3-deficient HSCs accumulate spontaneous DNA damage. (A–C) γH2AX immunostaining on sorted Lin − Sca1 + c-Kit + CD150 + flk2/CD135 − CD34 − (LT-HSC) or CD34 + (ST-HSC) from 44-wk-old (A and B) or 17-wk-old (C) mice. Representative images (A) of LT-HSCs and ST-HSCs from 44-wk-old mice and quantification of the number of γH2AX foci/cell in HSCs from 44-wk-old (B) or 17-wk-old mice (minimum of 200 cells per genotype; C). Results are from two independent experiments. n = 3 mice/genotype/experiment. Bar, 5 µm. (D–F) Alkaline comet assay on sorted Usp3 Δ/Δ LSKs (D and E) or total BM cells (F). Representative LSKs images (D) and the Average Tail Moment calculated by Comet Score on LSKs (E) or BM cells (F) are shown. A minimum of 150 comets was evaluated per sample. n = 3 per genotype, 44 wk old. Bar, 50 µm. (G–J) Sorted LT-HSCs from BM of 40–44 wk old mice were grown in liquid cultures and analyzed for DNA damage. (G) Immunostaining of γH2AX and 53BP1 on LT-HSCs after 8–11 d in culture. The percentage of cells containing > 5 γH2AX and 53BP1 foci was evaluated in three independent experiments. n > 50 cells/genotype/experiment. Arrows: γH2AX-53BP1 colocalizing foci. Bar, 5 µm. (H) Immunostaining of FK2 LT-HSCs after 8–11 d in culture. Representative images and quantification by Image J of FK2 signal intensity from three independent experiments. Nuclei are outlined. n > 100 cells/genotype/experiment. Bar, 10 µm. (I) Co-immunostaining of FK2 and 53BP1 on LT-HSCs after 8–11 d in culture. The number of co-foci (arrows) per cell was quantified in 2 independent experiments, on a total of n = 145 (WT) or 80 ( Usp3 Δ/Δ) cells scored. Bar, 10 µm. (J) Percentage of micronuclei in LT-HSCs cultures after 8 or 15 d in culture. A minimum of 70 cells/sample was scored in three independent experiments. Bars, 10 µm. In all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Results are mean ± SEM (B, E, F, H, and I) or mean ± SD (G and J).

    Techniques Used: Immunostaining, Mouse Assay, Alkaline Single Cell Gel Electrophoresis

    Reduced size of adult HSC and CLP compartments and impaired pre–B lymphoid colony-forming activity in vitro in Usp3 Δ/Δ mice. (A–C) Multiparameter flow cytometry analysis of primitive hematopoietic populations. Gating strategies and representative FACS profiles are presented in Fig. S2 . (A) Absolute cell numbers of primitive populations from BM (2 femurs and 2 hips bones) of WT and Usp3 Δ/Δ mice: LSK (Lin − Sca1 + cKit + ), LT-HSC (LSK, flk2/CD135 − , CD150 + , CD34 − , LT-HSC), and ST-HSC (LSK, flk2/CD135 − , CD150 + , CD34 + , ST-HSC). Mean ± SEM is shown. (B and C) Frequency of LSKs, LT-HSCs, ST-HSCs, and MPPs (B) or CLPs, CMPs, GMPs, and MEPs (C) in BM of Usp3 Δ/Δ mice was calculated and normalized relative to WT animals. Mean ± SD is shown. (A–C) Results are from two (17 wk) or three (44 wk) independent experiments. 17 wk, n = 5 per genotype; 44 wk, n = 11 per genotype. (D and E) BM cells from WT or Usp3 Δ/Δ mice were assayed for pre–B (D) or myeloid colony-forming (CFU-C; E) ability. Results are from at least two independent experiments, n = 3 per group per experiment. Mean ± SD is shown. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, not significant.
    Figure Legend Snippet: Reduced size of adult HSC and CLP compartments and impaired pre–B lymphoid colony-forming activity in vitro in Usp3 Δ/Δ mice. (A–C) Multiparameter flow cytometry analysis of primitive hematopoietic populations. Gating strategies and representative FACS profiles are presented in Fig. S2 . (A) Absolute cell numbers of primitive populations from BM (2 femurs and 2 hips bones) of WT and Usp3 Δ/Δ mice: LSK (Lin − Sca1 + cKit + ), LT-HSC (LSK, flk2/CD135 − , CD150 + , CD34 − , LT-HSC), and ST-HSC (LSK, flk2/CD135 − , CD150 + , CD34 + , ST-HSC). Mean ± SEM is shown. (B and C) Frequency of LSKs, LT-HSCs, ST-HSCs, and MPPs (B) or CLPs, CMPs, GMPs, and MEPs (C) in BM of Usp3 Δ/Δ mice was calculated and normalized relative to WT animals. Mean ± SD is shown. (A–C) Results are from two (17 wk) or three (44 wk) independent experiments. 17 wk, n = 5 per genotype; 44 wk, n = 11 per genotype. (D and E) BM cells from WT or Usp3 Δ/Δ mice were assayed for pre–B (D) or myeloid colony-forming (CFU-C; E) ability. Results are from at least two independent experiments, n = 3 per group per experiment. Mean ± SD is shown. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, not significant.

    Techniques Used: Activity Assay, In Vitro, Mouse Assay, Flow Cytometry, Cytometry, FACS

    Usp3 Δ/Δ mice exhibit shorter lifespan, increased tumorigenesis, and spontaneous genotoxic stress in MEFs. (A–E) Cohorts of WT ( n = 26) and Usp3 Δ/Δ ( n = 34) mice were monitored for survival for 90 wk. (A) Kaplan Meier general survival analysis. (B) Histopathological analysis of spleens from WT and Usp3 Δ/Δ mice and representative H E-stained spleen sections from 5-mo-old animals. Bars: (left) 500 µm; (right) 20 µm. a Low myelopoiesis in one 10-mo-old Usp3 Δ/Δ animal; b low lymphoid compartment in a 15-mo-old Usp3 Δ/Δ mouse. (C) Kaplan Meier tumor-free survival analysis and distribution of tumor types in Usp3 Δ/Δ mice. (D and E). H E staining of histological sections of representative malignancies in Usp3 Δ/Δ mice. (D) Moderately differentiated papillary carcinoma of the lung (17 mo). (E) Adenomatosis in the stomach (14 mo). Bars: (top) 500 µm; (bottom) 50 µm. (F) Constant field gel electrophoresis (CFGE) analysis of WT and Usp3 Δ/Δ MEFs. Results are the mean ± SD of three independent experiments. (G) Quantification of chromosomal aberrations in metaphase preparations of WT and Usp3 Δ/Δ MEF. A minimum of 42 cells/genotype was assessed. Mean ± SEM of one of two representative experiments is shown. (H) Metaphase analysis of WT and Usp3 Δ/Δ MEFs immortalized with p53 knockdown (sh-p53). Arrowheads: chromatid break, chromosome fragment, ring chromosome. Inset, chromatid break. Results are the mean ± SD of three independent experiments with a minimum of 30 metaphase/genotype each counted. Bar, 10 µm. (I) SCEs analysis in WT and Usp3 Δ/Δ sh-p53 MEFs. SCEs in representative metaphases are indicated by arrows. Inset, chromosome with double SCE. SCEs in a minimum of 48 cells/genotype were quantified. Mean ± SEM of one of two representative experiments is shown. Bar, 10 µm. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. P-value was assessed by Log-rank test (A and C) or by Student’s t test (F–I).
    Figure Legend Snippet: Usp3 Δ/Δ mice exhibit shorter lifespan, increased tumorigenesis, and spontaneous genotoxic stress in MEFs. (A–E) Cohorts of WT ( n = 26) and Usp3 Δ/Δ ( n = 34) mice were monitored for survival for 90 wk. (A) Kaplan Meier general survival analysis. (B) Histopathological analysis of spleens from WT and Usp3 Δ/Δ mice and representative H E-stained spleen sections from 5-mo-old animals. Bars: (left) 500 µm; (right) 20 µm. a Low myelopoiesis in one 10-mo-old Usp3 Δ/Δ animal; b low lymphoid compartment in a 15-mo-old Usp3 Δ/Δ mouse. (C) Kaplan Meier tumor-free survival analysis and distribution of tumor types in Usp3 Δ/Δ mice. (D and E). H E staining of histological sections of representative malignancies in Usp3 Δ/Δ mice. (D) Moderately differentiated papillary carcinoma of the lung (17 mo). (E) Adenomatosis in the stomach (14 mo). Bars: (top) 500 µm; (bottom) 50 µm. (F) Constant field gel electrophoresis (CFGE) analysis of WT and Usp3 Δ/Δ MEFs. Results are the mean ± SD of three independent experiments. (G) Quantification of chromosomal aberrations in metaphase preparations of WT and Usp3 Δ/Δ MEF. A minimum of 42 cells/genotype was assessed. Mean ± SEM of one of two representative experiments is shown. (H) Metaphase analysis of WT and Usp3 Δ/Δ MEFs immortalized with p53 knockdown (sh-p53). Arrowheads: chromatid break, chromosome fragment, ring chromosome. Inset, chromatid break. Results are the mean ± SD of three independent experiments with a minimum of 30 metaphase/genotype each counted. Bar, 10 µm. (I) SCEs analysis in WT and Usp3 Δ/Δ sh-p53 MEFs. SCEs in representative metaphases are indicated by arrows. Inset, chromosome with double SCE. SCEs in a minimum of 48 cells/genotype were quantified. Mean ± SEM of one of two representative experiments is shown. Bar, 10 µm. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. P-value was assessed by Log-rank test (A and C) or by Student’s t test (F–I).

    Techniques Used: Mouse Assay, Staining, Nucleic Acid Electrophoresis

    USP3-deficient mice develop lymphopenia with age. (A) Peripheral blood cell counts in aged (44 wk old) WT and Usp3 Δ/Δ mice. B220 + , B lymphocytes; CD3 + , T lymphocytes; CD11b + , monocytes, granulocytes, and macrophages. Data are means ± SD. WT, n = 7; Usp3 Δ/Δ, n = 7. Representative FACS profiles are shown in Fig. S1 . (B) Flow cytometry analysis of BM of aged WT and Usp3 Δ/Δ mice for lymphoid (CD19 +/low ) and myeloid (CD11b + ) cell populations. Cell numbers per BM (2 femurs) are shown. Data are means ± SEM. WT, n = 10; Usp3 Δ/Δ, n = 10. (C) Flow cytometry analysis of B cell differentiation in the BM of aged WT and Usp3 Δ/Δ mice: Pre–B (B220 low IgM − cKit − CD25 + ), Pro–B (B220 low IgM − cKit + CD25 − ), immature B (B220 low IgM + ), and mature B (B220 high IgM + ) cells. Cell numbers per BM (2 femurs) are shown. Data are means ± SD. WT, n = 8; Usp3 Δ/Δ, n = 7. (D) Frequency (percentage of total B220 + B cell population) of the B cell subsets analyzed in C. Results are from two (A, C, and D) or three (B) independent experiments. For all panels: **, P ≤ 0.01; ns, not significant.
    Figure Legend Snippet: USP3-deficient mice develop lymphopenia with age. (A) Peripheral blood cell counts in aged (44 wk old) WT and Usp3 Δ/Δ mice. B220 + , B lymphocytes; CD3 + , T lymphocytes; CD11b + , monocytes, granulocytes, and macrophages. Data are means ± SD. WT, n = 7; Usp3 Δ/Δ, n = 7. Representative FACS profiles are shown in Fig. S1 . (B) Flow cytometry analysis of BM of aged WT and Usp3 Δ/Δ mice for lymphoid (CD19 +/low ) and myeloid (CD11b + ) cell populations. Cell numbers per BM (2 femurs) are shown. Data are means ± SEM. WT, n = 10; Usp3 Δ/Δ, n = 10. (C) Flow cytometry analysis of B cell differentiation in the BM of aged WT and Usp3 Δ/Δ mice: Pre–B (B220 low IgM − cKit − CD25 + ), Pro–B (B220 low IgM − cKit + CD25 − ), immature B (B220 low IgM + ), and mature B (B220 high IgM + ) cells. Cell numbers per BM (2 femurs) are shown. Data are means ± SD. WT, n = 8; Usp3 Δ/Δ, n = 7. (D) Frequency (percentage of total B220 + B cell population) of the B cell subsets analyzed in C. Results are from two (A, C, and D) or three (B) independent experiments. For all panels: **, P ≤ 0.01; ns, not significant.

    Techniques Used: Mouse Assay, FACS, Flow Cytometry, Cytometry, Cell Differentiation

    Usp3 Δ/Δ mice are viable. (A) Generation of conditional ( Usp3 Lox ) and null ( Usp3 Δ) USP3 alleles. USP3 protein domains and gene locus are schematically represented. ZnF, zinc finger Ub binding domain (ZnF-UBP); USP, Ub-specific protease domain. The targeting construct for Usp3 (thick blue line) contains LoxP (L, red triangles) sites positioned in introns flanking exon 2 and 3. Numbered gray boxes: exons. Triangles: FRT (F) sites. Puro: puromycin Dtk selection cassette. Restriction enzymes used for screening: B, BamHI; E, EcoRI; K, KpnI. Thick black lines: DNA probes used in Southern blot analysis. (B) PCR analysis of genomic DNA isolated from targeted ES clones. (C–G) Actin-Cre deleter strain was used for germline deletion and intercrossing of Usp3 Δ/+ mice produced Usp3 Δ/Δ homozygous animals, confirmed by PCR analysis (C) and Southern blot (D) on tail tip DNA. (E) Genotype frequency per litter, on a total of 24 litters. n = number of born mice/genotype. Mean ± SD is shown. (F) Immunoblot of whole cell extract (WCE) from tissues from WT and Usp3 Δ/Δ mice with anti-USP3 and anti-CDK4 antibody. (G) Reverse transcription qPCR analysis of the relative expression of USP3 transcript in WT and Usp3 Δ/Δ MEFs (mouse embryonic fibroblasts).
    Figure Legend Snippet: Usp3 Δ/Δ mice are viable. (A) Generation of conditional ( Usp3 Lox ) and null ( Usp3 Δ) USP3 alleles. USP3 protein domains and gene locus are schematically represented. ZnF, zinc finger Ub binding domain (ZnF-UBP); USP, Ub-specific protease domain. The targeting construct for Usp3 (thick blue line) contains LoxP (L, red triangles) sites positioned in introns flanking exon 2 and 3. Numbered gray boxes: exons. Triangles: FRT (F) sites. Puro: puromycin Dtk selection cassette. Restriction enzymes used for screening: B, BamHI; E, EcoRI; K, KpnI. Thick black lines: DNA probes used in Southern blot analysis. (B) PCR analysis of genomic DNA isolated from targeted ES clones. (C–G) Actin-Cre deleter strain was used for germline deletion and intercrossing of Usp3 Δ/+ mice produced Usp3 Δ/Δ homozygous animals, confirmed by PCR analysis (C) and Southern blot (D) on tail tip DNA. (E) Genotype frequency per litter, on a total of 24 litters. n = number of born mice/genotype. Mean ± SD is shown. (F) Immunoblot of whole cell extract (WCE) from tissues from WT and Usp3 Δ/Δ mice with anti-USP3 and anti-CDK4 antibody. (G) Reverse transcription qPCR analysis of the relative expression of USP3 transcript in WT and Usp3 Δ/Δ MEFs (mouse embryonic fibroblasts).

    Techniques Used: Mouse Assay, Binding Assay, Construct, Selection, Southern Blot, Polymerase Chain Reaction, Isolation, Clone Assay, Produced, Real-time Polymerase Chain Reaction, Expressing

    USP3-deficient HSCs have a cell-autonomous defect in repopulating ability in vivo and in colony formation in vitro. (A) Competitive transplantation of BM cells from 8-wk-old WT or Usp3 Δ/Δ (CD45.2; test) mice with WT (CD45.1; support) BM cells showing total reconstitution (left) and contribution of donor-derived cells to B cell (B220 + ), T cell (CD3 + ), and myeloid (Gr1 + ) lineages (middle) in the blood, or to LSKs in the BM (right) of irradiated recipients at the indicated wpt. Data are mean ± SD ( n = 5 per genotype). One of two representative experiments is shown. PBC, peripheral blood cell. (B) Noncompetitive transplantation of BM cells from aged (39–42 wk old) WT or Usp3 Δ/Δ mice. Donor-derived Lin − , LSKs, and HSCs in primary recipients at 16 wpt is shown. Data are mean ± SD ( n = 5 per genotype). (C) WT or Usp3 Δ/Δ BM cells from 8-wk-old mice were used in noncompetitive serial transplantations. Donor-derived LSKs in the BM of secondary recipients (separated by a 12 wk reconstitution period) are shown. Data are mean ± SEM ( n = 5 per genotype). (D) Total BM cell numbers in WT or Usp3 Δ/Δ mice at 17 and 44 wk of age (WT = 5, Usp3 Δ/Δ = 6, in two independent experiments; 2 femurs and 2 hip bones) or in 44-wk-old mice ( n = 3 per genotype) upon 5-FU treatment (2 femurs). Data are mean ± SEM. (E) LTC-IC assay using WT or Usp3 Δ/Δ Lin − cells purified from 8–16-wk-old mice (three experiments, n = 4 mice/genotype/experiment). The number of LSKs in the Lin − populations was evaluated by phenotypic profiling before plating, and results are expressed as total number of CFU-C normalized to 2,000 LSK plated. Data are mean ± SEM. In all BM transplantations, BM cells corresponding to stem cell equivalents were transplanted. In B and C, BM cells from n = 3 donor mice per genotype were pooled before primary transplantation. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
    Figure Legend Snippet: USP3-deficient HSCs have a cell-autonomous defect in repopulating ability in vivo and in colony formation in vitro. (A) Competitive transplantation of BM cells from 8-wk-old WT or Usp3 Δ/Δ (CD45.2; test) mice with WT (CD45.1; support) BM cells showing total reconstitution (left) and contribution of donor-derived cells to B cell (B220 + ), T cell (CD3 + ), and myeloid (Gr1 + ) lineages (middle) in the blood, or to LSKs in the BM (right) of irradiated recipients at the indicated wpt. Data are mean ± SD ( n = 5 per genotype). One of two representative experiments is shown. PBC, peripheral blood cell. (B) Noncompetitive transplantation of BM cells from aged (39–42 wk old) WT or Usp3 Δ/Δ mice. Donor-derived Lin − , LSKs, and HSCs in primary recipients at 16 wpt is shown. Data are mean ± SD ( n = 5 per genotype). (C) WT or Usp3 Δ/Δ BM cells from 8-wk-old mice were used in noncompetitive serial transplantations. Donor-derived LSKs in the BM of secondary recipients (separated by a 12 wk reconstitution period) are shown. Data are mean ± SEM ( n = 5 per genotype). (D) Total BM cell numbers in WT or Usp3 Δ/Δ mice at 17 and 44 wk of age (WT = 5, Usp3 Δ/Δ = 6, in two independent experiments; 2 femurs and 2 hip bones) or in 44-wk-old mice ( n = 3 per genotype) upon 5-FU treatment (2 femurs). Data are mean ± SEM. (E) LTC-IC assay using WT or Usp3 Δ/Δ Lin − cells purified from 8–16-wk-old mice (three experiments, n = 4 mice/genotype/experiment). The number of LSKs in the Lin − populations was evaluated by phenotypic profiling before plating, and results are expressed as total number of CFU-C normalized to 2,000 LSK plated. Data are mean ± SEM. In all BM transplantations, BM cells corresponding to stem cell equivalents were transplanted. In B and C, BM cells from n = 3 donor mice per genotype were pooled before primary transplantation. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

    Techniques Used: In Vivo, In Vitro, Transplantation Assay, Mouse Assay, Derivative Assay, Irradiation, Purification

    5) Product Images from "Distinct BRCA1 Rearrangements Involving the BRCA1 Pseudogene Suggest the Existence of a Recombination Hot Spot"

    Article Title: Distinct BRCA1 Rearrangements Involving the BRCA1 Pseudogene Suggest the Existence of a Recombination Hot Spot

    Journal: American Journal of Human Genetics

    doi:

    BRCA1 color bar code of DNA samples from families F32 and F3514. Two allele populations could be visualized under a microscope when combed DNA samples were hybridized with fluorescent probes generated from PAC 103O14 ( green ) and cosmid D06121 ( red ) and from long-range PCR products NBR2 LR2-4 ( blue ), BRCA1 LR9-12 ( blue ), BRCA1 LR16-19 ( deep pink ), and BRCA1 LR24-3′ ( blue ). The sizes of the probes are indicated. The expected normal bar code for the wild-type allele was ΨBRCA1-NBR2-BRCA1 exons 1–2, 9–12, 16–19, and 24, as shown in A and C, for F32 and F3514, respectively. This pattern was found in 18 full signals (50%) for F32 and 26 full signals (58%) for F3514. An abnormal, shorter signal, without the 5′ part of the normal pattern, as shown in B and D, for F32 and F3514, respectively, was also found in 18 full signals (50%) for F32 and 19 full signals (42%) for F3514, corresponding to the mutant alleles with the deletion.
    Figure Legend Snippet: BRCA1 color bar code of DNA samples from families F32 and F3514. Two allele populations could be visualized under a microscope when combed DNA samples were hybridized with fluorescent probes generated from PAC 103O14 ( green ) and cosmid D06121 ( red ) and from long-range PCR products NBR2 LR2-4 ( blue ), BRCA1 LR9-12 ( blue ), BRCA1 LR16-19 ( deep pink ), and BRCA1 LR24-3′ ( blue ). The sizes of the probes are indicated. The expected normal bar code for the wild-type allele was ΨBRCA1-NBR2-BRCA1 exons 1–2, 9–12, 16–19, and 24, as shown in A and C, for F32 and F3514, respectively. This pattern was found in 18 full signals (50%) for F32 and 26 full signals (58%) for F3514. An abnormal, shorter signal, without the 5′ part of the normal pattern, as shown in B and D, for F32 and F3514, respectively, was also found in 18 full signals (50%) for F32 and 19 full signals (42%) for F3514, corresponding to the mutant alleles with the deletion.

    Techniques Used: Microscopy, Generated, Polymerase Chain Reaction, Mutagenesis

    6) Product Images from "Hemotin, a Regulator of Phagocytosis Encoded by a Small ORF and Conserved across Metazoans"

    Article Title: Hemotin, a Regulator of Phagocytosis Encoded by a Small ORF and Conserved across Metazoans

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1002395

    The Hemotin peptide is required for proper endosomal maturation in hemocytes. (A–A”) Distribution of acidic organelles in hemo A4 mutant ex vivo hemocytes revealed by the expression of LAMP1-GFP lysosomal marker. The intracellular vacuoles that disrupt the beta-tubulin cytoskeleton (A, A”; red) accumulate LAMP1-GFP positive compartments (A, A’; green). Compare with wild-type in S2A–S2A” Fig . Scale bar (5 μm). (B–B”) Distribution of the endosomal marker FYVE (PI(3)P binding zinc finger domain, early endosomal marker, named after being found in Fab1, YOTP, Vac1, EEA1) (green) (B, B’) and Lysotracker (red) (B, B”) organelles in a hemo A4 mutant ex vivo hemocyte showing enlarged intracellular compartments coexpressing FYVE and Lysotracker. Scale bar (5 μm). (C–C”) Wild-type ex vivo hemocyte labelled as in (B), showing little overlap between early endosome-FYVE positive (green) (C,C’) and lysosomal (red) (C,C”) compartments. (D) Quantification of the FYVE OAI in ex vivo hemocytes (see Materials and Methods ). hemo A4 mutants display a significantly larger FYVE area than wild-type. This phenotype is rescued by the expression of the hemo full length transcript ( UAS-hemoFL ) and is specific to hemo -ORF function, as it is also rescued by the expression of the hemo -ORF mini gene ( UAS-hemo-ORF ) or C-terminal-tagged hemo -GFP (UAS-hemo-GFP ). No rescue was observed by a CG7691 genomic fragment ( CG7691-GF ), or with a hemo full-length transcript containing a frameshift in the hemo -ORF ( UAS-hemoFS ), or with the ORF2 mini gene ( UAS-ORF2 ). All UAS constructs were driven with He-Gal4. Error bars represent SEM. One-way ANOVA analysis shows that there is a statistically significant difference between these groups [F(7,286) = 27.12, p
    Figure Legend Snippet: The Hemotin peptide is required for proper endosomal maturation in hemocytes. (A–A”) Distribution of acidic organelles in hemo A4 mutant ex vivo hemocytes revealed by the expression of LAMP1-GFP lysosomal marker. The intracellular vacuoles that disrupt the beta-tubulin cytoskeleton (A, A”; red) accumulate LAMP1-GFP positive compartments (A, A’; green). Compare with wild-type in S2A–S2A” Fig . Scale bar (5 μm). (B–B”) Distribution of the endosomal marker FYVE (PI(3)P binding zinc finger domain, early endosomal marker, named after being found in Fab1, YOTP, Vac1, EEA1) (green) (B, B’) and Lysotracker (red) (B, B”) organelles in a hemo A4 mutant ex vivo hemocyte showing enlarged intracellular compartments coexpressing FYVE and Lysotracker. Scale bar (5 μm). (C–C”) Wild-type ex vivo hemocyte labelled as in (B), showing little overlap between early endosome-FYVE positive (green) (C,C’) and lysosomal (red) (C,C”) compartments. (D) Quantification of the FYVE OAI in ex vivo hemocytes (see Materials and Methods ). hemo A4 mutants display a significantly larger FYVE area than wild-type. This phenotype is rescued by the expression of the hemo full length transcript ( UAS-hemoFL ) and is specific to hemo -ORF function, as it is also rescued by the expression of the hemo -ORF mini gene ( UAS-hemo-ORF ) or C-terminal-tagged hemo -GFP (UAS-hemo-GFP ). No rescue was observed by a CG7691 genomic fragment ( CG7691-GF ), or with a hemo full-length transcript containing a frameshift in the hemo -ORF ( UAS-hemoFS ), or with the ORF2 mini gene ( UAS-ORF2 ). All UAS constructs were driven with He-Gal4. Error bars represent SEM. One-way ANOVA analysis shows that there is a statistically significant difference between these groups [F(7,286) = 27.12, p

    Techniques Used: Mutagenesis, Ex Vivo, Expressing, Marker, Binding Assay, Construct

    Identification and phenotypical characterisation of the hemotin gene. (A) hemo genomic locus including the hemo , CG7691 , fray , and fruitless genes (blue arrows). The hemo A4 deletion (red bar) was generated by FRT-mediated recombination using the P{RS3}fray CB-0706-3 and the P-Bac{WH}fru f02684 transposable elements (blue triangles). Transcript models are represented under their respective genes, orange boxes represent coding exons, whereas gray boxes indicate noncoding exons (untranslated regions, UTRs). hemo A4 completely removes hemo and CG7691 plus the first noncoding exons of fray and fruitless . The P{PZ}fray 07551 insertion is a lethal fray allele [ 25 ] and was used for genetic complementation experiments between hemo A4 and fray . (B) Top: Ribosomal profiling reads obtained from polyribosomes from S2 cells (Poly-Riboseq;(3)) mapped to the hemo full-length transcript ( hemoFL ). hemo -ORF is translated more efficiently than ORF2 ( hemo -ORF RPKM: 29.4, coverage: 0.9 ORF; ORF2 RPKM: 6.6, coverage: 0.7. Note that the reads per kilobase of transcript per million mapped reads [RPKM] value of ORF2 is below the 11.8 cut-off to be considered translated [ 3 ]). Bottom: schematic representation of other constructs used in this manuscript. hemo -ORF is a minigene consisting of an mRNA fragment truncated immediately after the hemo -ORF stop codon, ORF2 consists of a mini-gene construct carrying the ORF2 sequence only, including 6 nt upstream of its start codon (to conserve its endogenous Kozak sequence). hemo -GFP (green fluorescent protein) is a hemo -ORF-GFP fusion construct in which the GFP sequence (devoid of a start codon) was cloned into the hemoFL construct, immediately downstream and in frame with hemo -ORF (devoid of a stop codon) (see Materials and Methods ). (C) Pattern of expression of hemo in germ band-retracted embryos revealed by in situ hybridisation. hemo is specifically expressed in embryonic hemocytes (arrows; compare with D) in the head, amnioserosa, and dispersed along the body. (D) Spatial distribution of embryonic hemocytes at germ band retraction stage revealed by in situ hybridisation of hemocyte-specific croquemort ( crq ) gene, showing similar distribution in the head, amnioserosa, and along the body (arrows). (E) Cluster of early embryonic hemocytes of the cephalic region expressing the hemo transcript revealed by FISH (fluorescent in situ hybridisation). Some hemocytes show drop-shape morphologies (asterisk) and membrane projections such as filopodia (arrows). (F) Embryonic hemocytes labelled with crq-Gal4;UAS-GFP expression from the head region displaying similar cellular morphologies (arrows and asterisk) as those in E. (G–H) White prepupal thoracic hemocytes revealed by crq-Gal4;UAS-GFP expression in wild-type (G) and hemo A4 mutants (H). In hemo A4 mutants, hemocytes display enlarged vacuoles within the cytoplasm (arrowheads), with larger occupied area index (OAI). Scale bar (50 μm). (I–N) hemocytes observed ex vivo [ 15 ] showing Tubulin (green) and Actin (red) cytoskeletons and nuclei (2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride, DAPI) with its corresponding orthogonal projection of confocal microscopy z-stacks (above inset) showing only tubulin cytoskeleton (green) and DAPI (blue) staining in the nucleus (n). Scale bar (5 μm). (I) Wild-type hemocyte. (J) hemo A4 mutant hemocyte shows large disruptions of the tubulin cytoskeleton that appear as rounded vacuoles (arrows; arrowhead in inset). (K) Knocking down the expression of hemo with a UAS-hemo-RNAi construct phenocopies the vacuolation phenotype (arrows and arrowhead in inset). (L) Expression of hemo full length transcript ( UAS-hemoFL ) rescues the vacuolated hemo A4 phenotype. Expression of hemo -ORF only (M) also rescues the hemo A4 mutant vacuolation. (N) Expression of ORF2 does not rescue the hemo A4 mutant vacuolated phenotype (arrows and arrowhead in inset). (O) Vacuolation measurements in ex vivo primary pre pupal hemocytes. hemo A4 mutant hemocytes show significantly higher occupied volume index (OVI) (see Materials and Methods ) than wild-type. Rescue experiments show that the vacuolation phenotype is specific to the peptide encoded by hemo -ORF. All upstream activating sequence (UAS) constructs were driven by crq-Gal4 . Error bars represent standard error of the mean (SEM). Statistical analysis was performed using one-way ANOVA test indicating that samples were significantly different [F(9,486) = 9.5, p
    Figure Legend Snippet: Identification and phenotypical characterisation of the hemotin gene. (A) hemo genomic locus including the hemo , CG7691 , fray , and fruitless genes (blue arrows). The hemo A4 deletion (red bar) was generated by FRT-mediated recombination using the P{RS3}fray CB-0706-3 and the P-Bac{WH}fru f02684 transposable elements (blue triangles). Transcript models are represented under their respective genes, orange boxes represent coding exons, whereas gray boxes indicate noncoding exons (untranslated regions, UTRs). hemo A4 completely removes hemo and CG7691 plus the first noncoding exons of fray and fruitless . The P{PZ}fray 07551 insertion is a lethal fray allele [ 25 ] and was used for genetic complementation experiments between hemo A4 and fray . (B) Top: Ribosomal profiling reads obtained from polyribosomes from S2 cells (Poly-Riboseq;(3)) mapped to the hemo full-length transcript ( hemoFL ). hemo -ORF is translated more efficiently than ORF2 ( hemo -ORF RPKM: 29.4, coverage: 0.9 ORF; ORF2 RPKM: 6.6, coverage: 0.7. Note that the reads per kilobase of transcript per million mapped reads [RPKM] value of ORF2 is below the 11.8 cut-off to be considered translated [ 3 ]). Bottom: schematic representation of other constructs used in this manuscript. hemo -ORF is a minigene consisting of an mRNA fragment truncated immediately after the hemo -ORF stop codon, ORF2 consists of a mini-gene construct carrying the ORF2 sequence only, including 6 nt upstream of its start codon (to conserve its endogenous Kozak sequence). hemo -GFP (green fluorescent protein) is a hemo -ORF-GFP fusion construct in which the GFP sequence (devoid of a start codon) was cloned into the hemoFL construct, immediately downstream and in frame with hemo -ORF (devoid of a stop codon) (see Materials and Methods ). (C) Pattern of expression of hemo in germ band-retracted embryos revealed by in situ hybridisation. hemo is specifically expressed in embryonic hemocytes (arrows; compare with D) in the head, amnioserosa, and dispersed along the body. (D) Spatial distribution of embryonic hemocytes at germ band retraction stage revealed by in situ hybridisation of hemocyte-specific croquemort ( crq ) gene, showing similar distribution in the head, amnioserosa, and along the body (arrows). (E) Cluster of early embryonic hemocytes of the cephalic region expressing the hemo transcript revealed by FISH (fluorescent in situ hybridisation). Some hemocytes show drop-shape morphologies (asterisk) and membrane projections such as filopodia (arrows). (F) Embryonic hemocytes labelled with crq-Gal4;UAS-GFP expression from the head region displaying similar cellular morphologies (arrows and asterisk) as those in E. (G–H) White prepupal thoracic hemocytes revealed by crq-Gal4;UAS-GFP expression in wild-type (G) and hemo A4 mutants (H). In hemo A4 mutants, hemocytes display enlarged vacuoles within the cytoplasm (arrowheads), with larger occupied area index (OAI). Scale bar (50 μm). (I–N) hemocytes observed ex vivo [ 15 ] showing Tubulin (green) and Actin (red) cytoskeletons and nuclei (2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride, DAPI) with its corresponding orthogonal projection of confocal microscopy z-stacks (above inset) showing only tubulin cytoskeleton (green) and DAPI (blue) staining in the nucleus (n). Scale bar (5 μm). (I) Wild-type hemocyte. (J) hemo A4 mutant hemocyte shows large disruptions of the tubulin cytoskeleton that appear as rounded vacuoles (arrows; arrowhead in inset). (K) Knocking down the expression of hemo with a UAS-hemo-RNAi construct phenocopies the vacuolation phenotype (arrows and arrowhead in inset). (L) Expression of hemo full length transcript ( UAS-hemoFL ) rescues the vacuolated hemo A4 phenotype. Expression of hemo -ORF only (M) also rescues the hemo A4 mutant vacuolation. (N) Expression of ORF2 does not rescue the hemo A4 mutant vacuolated phenotype (arrows and arrowhead in inset). (O) Vacuolation measurements in ex vivo primary pre pupal hemocytes. hemo A4 mutant hemocytes show significantly higher occupied volume index (OVI) (see Materials and Methods ) than wild-type. Rescue experiments show that the vacuolation phenotype is specific to the peptide encoded by hemo -ORF. All upstream activating sequence (UAS) constructs were driven by crq-Gal4 . Error bars represent standard error of the mean (SEM). Statistical analysis was performed using one-way ANOVA test indicating that samples were significantly different [F(9,486) = 9.5, p

    Techniques Used: Generated, BAC Assay, Construct, Sequencing, Clone Assay, Expressing, In Situ, Hybridization, Fluorescence In Situ Hybridization, Ex Vivo, Confocal Microscopy, Staining, Mutagenesis

    7) Product Images from "Effect of Leflunomide, Cidofovir and Ciprofloxacin on Replication of BKPyV in a Salivary Gland In Vitro Culture System"

    Article Title: Effect of Leflunomide, Cidofovir and Ciprofloxacin on Replication of BKPyV in a Salivary Gland In Vitro Culture System

    Journal: Antiviral research

    doi: 10.1016/j.antiviral.2015.02.002

    Effect of drug treatment on lab-strain and patient-derived BKPy virus progeny release from human salivary gland cells HSG cells were infected with ( A ) lab-strain BKPyV (VR837) or ( B ) BKPyV isolated from the saliva of two HIVSGD patients and a lab-adapted virus strain (MM), or (C) BKPyV isolated from urine of a lung transplant patient, and treated with drug as described in the materials and methods. At stated times post infection supernatant was collected, Dnase-treated and qPCR performed for TAg and VP1 (data not shown) DNA copy no. A standard curve (data not shown) was constructed using a plasmid coding for BKPyV whole genome. The error bars represent the SD and p-value calculated using the t-test.
    Figure Legend Snippet: Effect of drug treatment on lab-strain and patient-derived BKPy virus progeny release from human salivary gland cells HSG cells were infected with ( A ) lab-strain BKPyV (VR837) or ( B ) BKPyV isolated from the saliva of two HIVSGD patients and a lab-adapted virus strain (MM), or (C) BKPyV isolated from urine of a lung transplant patient, and treated with drug as described in the materials and methods. At stated times post infection supernatant was collected, Dnase-treated and qPCR performed for TAg and VP1 (data not shown) DNA copy no. A standard curve (data not shown) was constructed using a plasmid coding for BKPyV whole genome. The error bars represent the SD and p-value calculated using the t-test.

    Techniques Used: Derivative Assay, Infection, Isolation, Real-time Polymerase Chain Reaction, Construct, Plasmid Preparation

    8) Product Images from "Distinct BRCA1 Rearrangements Involving the BRCA1 Pseudogene Suggest the Existence of a Recombination Hot Spot"

    Article Title: Distinct BRCA1 Rearrangements Involving the BRCA1 Pseudogene Suggest the Existence of a Recombination Hot Spot

    Journal: American Journal of Human Genetics

    doi:

    Representation of the 37-kb deletion in F32 and F3514. Drawn-to-scale schema of the normal and mutant alleles at the NBR1 and BRCA1 loci, showing the location of the 36,934-bp deletion. The size of the NBR1, ΨBRCA1, NBR2, and BRCA1 genes are given in parentheses in the normal allele schema.
    Figure Legend Snippet: Representation of the 37-kb deletion in F32 and F3514. Drawn-to-scale schema of the normal and mutant alleles at the NBR1 and BRCA1 loci, showing the location of the 36,934-bp deletion. The size of the NBR1, ΨBRCA1, NBR2, and BRCA1 genes are given in parentheses in the normal allele schema.

    Techniques Used: Mutagenesis

    BRCA1 color bar code of DNA samples from families F32 and F3514. Two allele populations could be visualized under a microscope when combed DNA samples were hybridized with fluorescent probes generated from PAC 103O14 ( green ) and cosmid D06121 ( red ) and from long-range PCR products NBR2 LR2-4 ( blue ), BRCA1 LR9-12 ( blue ), BRCA1 LR16-19 ( deep pink ), and BRCA1 LR24-3′ ( blue ). The sizes of the probes are indicated. The expected normal bar code for the wild-type allele was ΨBRCA1-NBR2-BRCA1 exons 1–2, 9–12, 16–19, and 24, as shown in A and C, for F32 and F3514, respectively. This pattern was found in 18 full signals (50%) for F32 and 26 full signals (58%) for F3514. An abnormal, shorter signal, without the 5′ part of the normal pattern, as shown in B and D, for F32 and F3514, respectively, was also found in 18 full signals (50%) for F32 and 19 full signals (42%) for F3514, corresponding to the mutant alleles with the deletion.
    Figure Legend Snippet: BRCA1 color bar code of DNA samples from families F32 and F3514. Two allele populations could be visualized under a microscope when combed DNA samples were hybridized with fluorescent probes generated from PAC 103O14 ( green ) and cosmid D06121 ( red ) and from long-range PCR products NBR2 LR2-4 ( blue ), BRCA1 LR9-12 ( blue ), BRCA1 LR16-19 ( deep pink ), and BRCA1 LR24-3′ ( blue ). The sizes of the probes are indicated. The expected normal bar code for the wild-type allele was ΨBRCA1-NBR2-BRCA1 exons 1–2, 9–12, 16–19, and 24, as shown in A and C, for F32 and F3514, respectively. This pattern was found in 18 full signals (50%) for F32 and 26 full signals (58%) for F3514. An abnormal, shorter signal, without the 5′ part of the normal pattern, as shown in B and D, for F32 and F3514, respectively, was also found in 18 full signals (50%) for F32 and 19 full signals (42%) for F3514, corresponding to the mutant alleles with the deletion.

    Techniques Used: Microscopy, Generated, Polymerase Chain Reaction, Mutagenesis

    9) Product Images from "Expression of a large LINE-1-driven antisense RNA is linked to epigenetic silencing of the metastasis suppressor gene TFPI-2 in cancer"

    Article Title: Expression of a large LINE-1-driven antisense RNA is linked to epigenetic silencing of the metastasis suppressor gene TFPI-2 in cancer

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt438

    A human TFPI-2 transgene is sensitive to antisense RNA repression in mouse ES cells. (A) Schematic diagram of constructs introduced into mouse ES cells: pTFPI-2as is designed to transcribe antisense to TFPI-2 from a CMV promoter, while pTFPI-2pa has a poly-A signal insertion downstream of the CMV promoter to block antisense transcription. Arrows indicate direction of transcription. Regions analysed by ChIP are annotated as ‘prom’ and ‘ex-in2’. ( B ) Strand-specific RT–PCR analysis of TFPI-2 antisenese (TFPI-2as) expression in transgenic mouse ES cell lines demonstrates increased levels in pTFPI-2as lines (L2 and L12) relative to pTFPI-2pa cells (L7 and L9), mouse Aprt acts as a positive control for RNA quality and quantity. This correlates with a reduction in TFPI-2 expression as shown by real-time PCR normalized to mouse Gapdh . ( C ) ChIP analysis followed by real-time PCR. Left panel: Antibodies to H3K9me3 reveal localized enrichment of H3K9me3 in the promoter region in the antisense expressing cell line, pTFPI-2as (L2), compared to cells transfected with pTFPI-2pa (L9), which express low levels of TFPI-2as. Right panel: Antibodies to H4K20me3 also show enrichment at the TFPI-2 promoter in pTFPI-2as compared to pTFPI-2pa.
    Figure Legend Snippet: A human TFPI-2 transgene is sensitive to antisense RNA repression in mouse ES cells. (A) Schematic diagram of constructs introduced into mouse ES cells: pTFPI-2as is designed to transcribe antisense to TFPI-2 from a CMV promoter, while pTFPI-2pa has a poly-A signal insertion downstream of the CMV promoter to block antisense transcription. Arrows indicate direction of transcription. Regions analysed by ChIP are annotated as ‘prom’ and ‘ex-in2’. ( B ) Strand-specific RT–PCR analysis of TFPI-2 antisenese (TFPI-2as) expression in transgenic mouse ES cell lines demonstrates increased levels in pTFPI-2as lines (L2 and L12) relative to pTFPI-2pa cells (L7 and L9), mouse Aprt acts as a positive control for RNA quality and quantity. This correlates with a reduction in TFPI-2 expression as shown by real-time PCR normalized to mouse Gapdh . ( C ) ChIP analysis followed by real-time PCR. Left panel: Antibodies to H3K9me3 reveal localized enrichment of H3K9me3 in the promoter region in the antisense expressing cell line, pTFPI-2as (L2), compared to cells transfected with pTFPI-2pa (L9), which express low levels of TFPI-2as. Right panel: Antibodies to H4K20me3 also show enrichment at the TFPI-2 promoter in pTFPI-2as compared to pTFPI-2pa.

    Techniques Used: Construct, Blocking Assay, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Expressing, Transgenic Assay, Positive Control, Real-time Polymerase Chain Reaction, Transfection

    10) Product Images from "Loss of Hippocampal Serine Protease BSP1/Neuropsin Predisposes to Global Seizure Activity"

    Article Title: Loss of Hippocampal Serine Protease BSP1/Neuropsin Predisposes to Global Seizure Activity

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.21-18-06993.2001

    Immediate early (c-fos) gene expression and physiological response to kainic acid challenge. A , c-fos mRNA expression revealed by in situ hybridization in BSP1/neuropsin knock-out (−/−) animals ( bottom ) and wild-type littermate controls (+/+; top ). Example expression patterns are shown before (0) and 120 min after injection of kainic acid (15 mg/kg, i.p.); increased expression is seen in BSP1/neuropsin knock-out mice. B , Survival of mice after injection of kainic acid (30 mg/kg, i.p.). BSP1/neuropsin homozygous knock-out animals (−/−; dashed line ; n = 8) display significant susceptibility compared with wild-type littermate controls (+/+; solid line ; n = 10). C , c-fos mRNA expression ( in situ hybridization) in BSP1/neuropsin knock-out (−/−) animals ( bottom ) and wild-type littermate controls (+/+; top ) at various time points (in minutes) after injection of kainic acid (30 mg/kg, i.p.). Induction of increased expression is demonstrated in BSP1/neuropsin mutant animals at all time points.
    Figure Legend Snippet: Immediate early (c-fos) gene expression and physiological response to kainic acid challenge. A , c-fos mRNA expression revealed by in situ hybridization in BSP1/neuropsin knock-out (−/−) animals ( bottom ) and wild-type littermate controls (+/+; top ). Example expression patterns are shown before (0) and 120 min after injection of kainic acid (15 mg/kg, i.p.); increased expression is seen in BSP1/neuropsin knock-out mice. B , Survival of mice after injection of kainic acid (30 mg/kg, i.p.). BSP1/neuropsin homozygous knock-out animals (−/−; dashed line ; n = 8) display significant susceptibility compared with wild-type littermate controls (+/+; solid line ; n = 10). C , c-fos mRNA expression ( in situ hybridization) in BSP1/neuropsin knock-out (−/−) animals ( bottom ) and wild-type littermate controls (+/+; top ) at various time points (in minutes) after injection of kainic acid (30 mg/kg, i.p.). Induction of increased expression is demonstrated in BSP1/neuropsin mutant animals at all time points.

    Techniques Used: Expressing, In Situ Hybridization, Knock-Out, Injection, Mouse Assay, Mutagenesis

    Baseline synaptic transmission in BSP1/neuropsin mutant animals. A , Peak-evoked fEPSP amplitudes in response to single-shock stimulation. Amplitudes over a range of stimulus intensities are plotted against the peak amplitudes of the presynaptic fiber volleys ( PSFV ) immediately preceding the fEPSPs for both wild-type and BSP1/neuropsin −/− slices ( n = 8 for each genotype). B, Left , Population spikes in wild-type ( top ) and BSP1/neuropsin −/− ( bottom ) slices. Right , Graph depicting the pooled maximum peak amplitude of these responses sampled ( n = 6 in each case) from wild-type and BSP1 −/− animals. C, Left , Synaptic traces showing fEPSPs recorded in response to paired-pulse stimulation delivered at an interstimulus interval of 25 msec in control ( top ) or BSP1/neuropsin −/− ( bottom ) slices. Right , Graph recording the magnitude and temporal profile of paired-pulse facilitation (calculated as the slope of the second fEPSP divided by the slope of the first EPSP) in wild-type and BSP1−/− slices ( n = 8 for each genotype).
    Figure Legend Snippet: Baseline synaptic transmission in BSP1/neuropsin mutant animals. A , Peak-evoked fEPSP amplitudes in response to single-shock stimulation. Amplitudes over a range of stimulus intensities are plotted against the peak amplitudes of the presynaptic fiber volleys ( PSFV ) immediately preceding the fEPSPs for both wild-type and BSP1/neuropsin −/− slices ( n = 8 for each genotype). B, Left , Population spikes in wild-type ( top ) and BSP1/neuropsin −/− ( bottom ) slices. Right , Graph depicting the pooled maximum peak amplitude of these responses sampled ( n = 6 in each case) from wild-type and BSP1 −/− animals. C, Left , Synaptic traces showing fEPSPs recorded in response to paired-pulse stimulation delivered at an interstimulus interval of 25 msec in control ( top ) or BSP1/neuropsin −/− ( bottom ) slices. Right , Graph recording the magnitude and temporal profile of paired-pulse facilitation (calculated as the slope of the second fEPSP divided by the slope of the first EPSP) in wild-type and BSP1−/− slices ( n = 8 for each genotype).

    Techniques Used: Transmission Assay, Mutagenesis

    LTP in BSP1/neuropsin mutant and control mice. Graph showing the field EPSP slope expressed as a percentage of control responses over time. All data points represent 5–10 averaged fEPSPs; error bars indicate SEM. At time 0, four trains of stimulation (100 shocks at 100 Hz; interstimulus interval, 5 min) were delivered to the Schaffer collateral–commissural pathway. Closed circles represent pooled data from wild-type control mice, and open circles represent BSP1/neuropsin mutant pooled data. Insets , Synaptic traces representing two superimposed field EPSPs recorded in the stratum radiatum of area CA1 in response to single-shock stimulation in the same dendritic field immediately before tetanic stimulation and 3 hr after conditioning in hippocampal slices taken from wild-type ( left ) and BSP1/neuropsin −/− ( right ) mice.
    Figure Legend Snippet: LTP in BSP1/neuropsin mutant and control mice. Graph showing the field EPSP slope expressed as a percentage of control responses over time. All data points represent 5–10 averaged fEPSPs; error bars indicate SEM. At time 0, four trains of stimulation (100 shocks at 100 Hz; interstimulus interval, 5 min) were delivered to the Schaffer collateral–commissural pathway. Closed circles represent pooled data from wild-type control mice, and open circles represent BSP1/neuropsin mutant pooled data. Insets , Synaptic traces representing two superimposed field EPSPs recorded in the stratum radiatum of area CA1 in response to single-shock stimulation in the same dendritic field immediately before tetanic stimulation and 3 hr after conditioning in hippocampal slices taken from wild-type ( left ) and BSP1/neuropsin −/− ( right ) mice.

    Techniques Used: Mutagenesis, Mouse Assay

    Targeted disruption of the murine BSP1/neuropsin locus. A , Structure of the targeting vector ( top ), wild-type BSP1/neuropsin allele ( middle ), and targeted allele ( bottom ) showing deletion of the catalytic aspartic acid residue region of exon III; asterisks indicate active site residues. A selection cassette (MC1–neo–pA), conferring resistance to G418 and a reporter cassette (IRES–lacZ–pA) allowing dicistronic translation of the bacterial β-galactosidase gene from the targeted allele were inserted. Two copies of a herpes simplex virus thymidine kinase gene (MC1–tk) were included in the vector for selection against nonhomologous integration. BSP1/neuropsin coding regions are indicated by filled boxes ; the position of Eco RV ( E ) and Hind III ( H ) restriction sites and the probe used for the 5′ targeting screen are shown. B , Southern blot analysis of tail genomic DNA from wild-type (+/+), heterozygous (+/−), and homozygous knock-out (−/−) animals demonstrating disruption of the BPS1/neuropsin gene. The probe hybridizes to a 3.1 kb Eco RV wild-type fragment and a 1.8 kb Eco RV fragment from the targeted allele. C, Top , Northern blot analysis of total RNA of the hippocampus ( Hi ) and rest of the brain ( R ) from BSP1/neuropsin mutant animals (−/−) and wild-type littermates (+/+) hybridized with the BSP1/neuropsin cDNA. Bottom , The same blot hybridized with S26 ribosomal protein cDNA demonstrating equal loading of the blot. D , Photomicrograph of cresyl violet-stained horizontal sections through the hippocampus and entorhinal cortex of wild-type (+/+; left ) and BSP1/neuropsin mutant (−/−; right ) animals.
    Figure Legend Snippet: Targeted disruption of the murine BSP1/neuropsin locus. A , Structure of the targeting vector ( top ), wild-type BSP1/neuropsin allele ( middle ), and targeted allele ( bottom ) showing deletion of the catalytic aspartic acid residue region of exon III; asterisks indicate active site residues. A selection cassette (MC1–neo–pA), conferring resistance to G418 and a reporter cassette (IRES–lacZ–pA) allowing dicistronic translation of the bacterial β-galactosidase gene from the targeted allele were inserted. Two copies of a herpes simplex virus thymidine kinase gene (MC1–tk) were included in the vector for selection against nonhomologous integration. BSP1/neuropsin coding regions are indicated by filled boxes ; the position of Eco RV ( E ) and Hind III ( H ) restriction sites and the probe used for the 5′ targeting screen are shown. B , Southern blot analysis of tail genomic DNA from wild-type (+/+), heterozygous (+/−), and homozygous knock-out (−/−) animals demonstrating disruption of the BPS1/neuropsin gene. The probe hybridizes to a 3.1 kb Eco RV wild-type fragment and a 1.8 kb Eco RV fragment from the targeted allele. C, Top , Northern blot analysis of total RNA of the hippocampus ( Hi ) and rest of the brain ( R ) from BSP1/neuropsin mutant animals (−/−) and wild-type littermates (+/+) hybridized with the BSP1/neuropsin cDNA. Bottom , The same blot hybridized with S26 ribosomal protein cDNA demonstrating equal loading of the blot. D , Photomicrograph of cresyl violet-stained horizontal sections through the hippocampus and entorhinal cortex of wild-type (+/+; left ) and BSP1/neuropsin mutant (−/−; right ) animals.

    Techniques Used: Plasmid Preparation, Selection, Southern Blot, Knock-Out, Northern Blot, Mutagenesis, Staining

    Spatial reference memory in BSP1/neuropsin mutant and control mice. A , Graph showing the mean latency of escape of BSP1/neuropsin mutant mice ( open circles ; n = 8) and wild-type littermate controls ( closed circles ; n = 8) against trial day. Both groups show a significant interaction between latency and trial day, indicating learning (ANOVA, p
    Figure Legend Snippet: Spatial reference memory in BSP1/neuropsin mutant and control mice. A , Graph showing the mean latency of escape of BSP1/neuropsin mutant mice ( open circles ; n = 8) and wild-type littermate controls ( closed circles ; n = 8) against trial day. Both groups show a significant interaction between latency and trial day, indicating learning (ANOVA, p

    Techniques Used: Mutagenesis, Mouse Assay

    11) Product Images from "Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare"

    Article Title: Large Interruptions of GAA Repeat Expansion Mutations in Friedreich Ataxia Are Very Rare

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2018.00443

    Mbo II digest results. Agarose gel showing Mbo II digests of GAA PCR products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.
    Figure Legend Snippet: Mbo II digest results. Agarose gel showing Mbo II digests of GAA PCR products of FRDA samples. The expected 170bp (5′) and 120bp (3′) undigested GAA-flanking fragments from normal pure GAA repeat expansion FRDA samples are shown in lanes 2, 3, and 4. These band sizes can be seen in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder markers, which are loaded into lanes 1 and 11 of the gel. Lane 5 shows a large Mbo II band of approximately 600bp that was obtained from the positive interrupted GAA repeat sequence from the “NEP” BAC transgenic mouse that contains approximately 500 triplet repeats with the previously determined interrupted sequence of (GAA) 21 (GGAGAA) 5 (GGAGGAGAA) 70 (GAA) n ). In addition for this positive sample, we also identified the expected 5′ flanking band of 170bp, together with a smaller band of less than 100bp that we sequenced and we showed to contain a 27bp deletion in the 3′ flanking region. Lane 6 shows an abnormal band of 200bp representing the 80bp duplication in the 3′ GAA flanking region. Lane 7 shows an abnormal band of approximately 100bp representing the 19bp deletion in the 3′ GAA flanking region. Lanes 8, 9, and 10 contain abnormal bands of approximately 300, 100, and 180bp, respectively, that are likely to contain a region of interrupted GAA repeat sequence within the body of one or other of the large FRDA GAA repeat expansions.

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Sequencing, BAC Assay, Transgenic Assay

    Mbo II digests of GAA repeat expansions from human FRDA somatic tissues and mouse FRDA intergenerational and somatic tissues. Agarose gels showing Mbo II digests of GAA PCR products of (A) FRDA patient cerebellum tissue samples, (B) YG8sR mouse ear biopsy samples and human FRDA blood samples, and (C) four tissues from one YG8sR mouse. In each case, the expected 170 and 120bp undigested GAA-flanking fragments can be identified in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder marker, which is loaded into the first lane of each gel. (A) Lanes 1–3 show the results from cerebellum tissue samples from three FRDA patients. (B) Lanes 1 and 2 are from FRDA patient blood samples; lanes 3–6 are from ear biopsy samples from 4 GAA repeat expansion-based YG8sR mice of four different generations, and lane 7 is from an ear biopsy sample from the Y47R mouse which has nine GAA repeats. (C) Lanes 1–4 are from brain, cerebellum, heart, and liver tissues of the YG8sR mouse, respectively.
    Figure Legend Snippet: Mbo II digests of GAA repeat expansions from human FRDA somatic tissues and mouse FRDA intergenerational and somatic tissues. Agarose gels showing Mbo II digests of GAA PCR products of (A) FRDA patient cerebellum tissue samples, (B) YG8sR mouse ear biopsy samples and human FRDA blood samples, and (C) four tissues from one YG8sR mouse. In each case, the expected 170 and 120bp undigested GAA-flanking fragments can be identified in between the 200 and 100bp fragments of the 1 Kb+ DNA ladder marker, which is loaded into the first lane of each gel. (A) Lanes 1–3 show the results from cerebellum tissue samples from three FRDA patients. (B) Lanes 1 and 2 are from FRDA patient blood samples; lanes 3–6 are from ear biopsy samples from 4 GAA repeat expansion-based YG8sR mice of four different generations, and lane 7 is from an ear biopsy sample from the Y47R mouse which has nine GAA repeats. (C) Lanes 1–4 are from brain, cerebellum, heart, and liver tissues of the YG8sR mouse, respectively.

    Techniques Used: Polymerase Chain Reaction, Marker, Mouse Assay

    12) Product Images from "A large deletion in RPGR causes XLPRA in Weimaraner dogs"

    Article Title: A large deletion in RPGR causes XLPRA in Weimaraner dogs

    Journal: Canine Genetics and Epidemiology

    doi: 10.1186/s40575-016-0037-x

    The deletion in the X chromosomal RPGR gene as identified in a PRA pedigree of Weimaraner dogs via whole exome sequencing; the breakpoint (BP) region is indicated. a Pedigree structure and RPGR deletion genotypes of 18 investigated individuals of the XLPRA Weimaraner family. PRA segregates in two generations of the family. Squares represent males, circles indicate females, crossed-out symbols represent deceased dogs. Filled squares show ophthalmologically diagnosed PRA-affected male dogs. Half-filled circles indicate females with ophthalmologically confirmed mild PRA symptoms. Open symbols represent male and female dogs with normal sight as revealed by general veterinarian examinations, respectively. An asterisk below solid square symbols indicates PRA-diagnosed dogs, which were used for whole exome sequencing. Genotypes of RPGR deletion screening are shown below the symbols. X M (colored in red) refers to an allele with RPGR deletion, X and Y symbols illustrate normal X- (with wildtype RPGR alleles) and Y-chromosomes, respectively. b Integrated Genomics Viewer (IGV) display of the canine RPGR deletion and surrounding regions (CFAX: 3310100–33106500, CanFam3.1 UCSC genome browser) as well as graphical illustration of exon-intron boundaries from the 5′UTR to exon 5. As viewed in IGV, the control and male PRA-affected dog are represented by two separate panels. The upper panel is a histogram where the height of each mountain-like grey area is representative of the read depth at that location. The lower panel is a graphical view of some of the reads that align to that location. Lack of reads (horizontal bars in lower panel) is characteristic for complete loss of exonic sequences. The deletion comprising exons 1–4 (~5 kb) is obvious in the male PRA-affected dog in hemizygous state in contrast to the PRA-unaffected dog. Thus the gap region includes exon 1 in the canine genomic RPGR sequence explaining only non-specific read alignments in the lower panel for the control. c QPCR-based copy number analysis of the deleted exons 3–4 and the non-deleted exon 5 of RPGR gene in four individuals of the pedigree of Weimaraners in comparison to a healthy control. Error bars indicate the standard deviation of three replicates. d Chromatogram and graphical representation of the BP region in the RPGR gene in a male PRA-affected Weimaraner. The graphical illustration indicates part of intron 4 sequence and of 5′UTR of RPGR . Deleted sequences of intron 4 and 5′UTR are coloured in light grey, non-deleted sequences are indicated by coloured letters. The BP region comprises three nucleotides (TTC) from either end which are underlined. The chromatogram also shows the BP (marked with arrows) as well as flanking sequences of intron 4 and 5′UTR of RPGR as identified in a male PRA-affected Weimaraner
    Figure Legend Snippet: The deletion in the X chromosomal RPGR gene as identified in a PRA pedigree of Weimaraner dogs via whole exome sequencing; the breakpoint (BP) region is indicated. a Pedigree structure and RPGR deletion genotypes of 18 investigated individuals of the XLPRA Weimaraner family. PRA segregates in two generations of the family. Squares represent males, circles indicate females, crossed-out symbols represent deceased dogs. Filled squares show ophthalmologically diagnosed PRA-affected male dogs. Half-filled circles indicate females with ophthalmologically confirmed mild PRA symptoms. Open symbols represent male and female dogs with normal sight as revealed by general veterinarian examinations, respectively. An asterisk below solid square symbols indicates PRA-diagnosed dogs, which were used for whole exome sequencing. Genotypes of RPGR deletion screening are shown below the symbols. X M (colored in red) refers to an allele with RPGR deletion, X and Y symbols illustrate normal X- (with wildtype RPGR alleles) and Y-chromosomes, respectively. b Integrated Genomics Viewer (IGV) display of the canine RPGR deletion and surrounding regions (CFAX: 3310100–33106500, CanFam3.1 UCSC genome browser) as well as graphical illustration of exon-intron boundaries from the 5′UTR to exon 5. As viewed in IGV, the control and male PRA-affected dog are represented by two separate panels. The upper panel is a histogram where the height of each mountain-like grey area is representative of the read depth at that location. The lower panel is a graphical view of some of the reads that align to that location. Lack of reads (horizontal bars in lower panel) is characteristic for complete loss of exonic sequences. The deletion comprising exons 1–4 (~5 kb) is obvious in the male PRA-affected dog in hemizygous state in contrast to the PRA-unaffected dog. Thus the gap region includes exon 1 in the canine genomic RPGR sequence explaining only non-specific read alignments in the lower panel for the control. c QPCR-based copy number analysis of the deleted exons 3–4 and the non-deleted exon 5 of RPGR gene in four individuals of the pedigree of Weimaraners in comparison to a healthy control. Error bars indicate the standard deviation of three replicates. d Chromatogram and graphical representation of the BP region in the RPGR gene in a male PRA-affected Weimaraner. The graphical illustration indicates part of intron 4 sequence and of 5′UTR of RPGR . Deleted sequences of intron 4 and 5′UTR are coloured in light grey, non-deleted sequences are indicated by coloured letters. The BP region comprises three nucleotides (TTC) from either end which are underlined. The chromatogram also shows the BP (marked with arrows) as well as flanking sequences of intron 4 and 5′UTR of RPGR as identified in a male PRA-affected Weimaraner

    Techniques Used: Sequencing, Real-time Polymerase Chain Reaction, Standard Deviation

    13) Product Images from "Expansion of a novel endogenous retrovirus throughout the pericentromeres of modern humans"

    Article Title: Expansion of a novel endogenous retrovirus throughout the pericentromeres of modern humans

    Journal: Genome Biology

    doi: 10.1186/s13059-015-0641-1

    Genomic structure and nucleotide differences of full-length K111, K222, and K222/K111 recombinant proviruses.  (A)  Highlighter plot showing the nucleotide differences between K111 along with K222 provirus found in a WGS database (Acc. No. AADC01167561.1) and K222/K111 recombinant provirus isolated from the genome of the H9 cell line indicated by tick marks (green ticks: A; red ticks: T; orange ticks: G; light blue ticks: C). Gray boxes denote areas deleted in K222.  (B)  Recombination plot of K222/K111 provirus. The similarity between the query K222/K111 recombinant sequence and each parental K222 and K111 provirus is plotted for each position of an approximately 10 Kb bp sliding window. The Y axis represents the match fraction of the query sequence to each parental sequence (red and blue lines). A match fraction of 1 means 100% identity. The recombinant query sequence is illustrated on the X axis (upper red/blue line at the top). Arrows indicate recombination spots.  (C)  A phylogenetic dendrogram displays three major clades; the 3′ LTR K111 (sometimes called K105) sequences previously reported (10; black), 3′ LTR K222 sequences found in human databases (blue), and the 3′ LTR of K222 sequences found in H9 and HUT78 cell lines (yellow). Previous sequences assigned by us as K105J and K105K were indeed K222 sequences and were flanked by pCER:D22Z8 repeat.  (D)  K222 and K111 proviruses arose by independent infections. A Bayesian inference tree shows the clustering of the 5′ and 3′ LTRs from various HERV-K (HML-2) proviruses. The K111 5′ LTR (red) and the 3′ LTRs of K111 (blue) and K222 (gray) proviruses cluster in three independent clades with a common ancestor. Posterior probability values  >  70 are shown.
    Figure Legend Snippet: Genomic structure and nucleotide differences of full-length K111, K222, and K222/K111 recombinant proviruses. (A) Highlighter plot showing the nucleotide differences between K111 along with K222 provirus found in a WGS database (Acc. No. AADC01167561.1) and K222/K111 recombinant provirus isolated from the genome of the H9 cell line indicated by tick marks (green ticks: A; red ticks: T; orange ticks: G; light blue ticks: C). Gray boxes denote areas deleted in K222. (B) Recombination plot of K222/K111 provirus. The similarity between the query K222/K111 recombinant sequence and each parental K222 and K111 provirus is plotted for each position of an approximately 10 Kb bp sliding window. The Y axis represents the match fraction of the query sequence to each parental sequence (red and blue lines). A match fraction of 1 means 100% identity. The recombinant query sequence is illustrated on the X axis (upper red/blue line at the top). Arrows indicate recombination spots. (C) A phylogenetic dendrogram displays three major clades; the 3′ LTR K111 (sometimes called K105) sequences previously reported (10; black), 3′ LTR K222 sequences found in human databases (blue), and the 3′ LTR of K222 sequences found in H9 and HUT78 cell lines (yellow). Previous sequences assigned by us as K105J and K105K were indeed K222 sequences and were flanked by pCER:D22Z8 repeat. (D) K222 and K111 proviruses arose by independent infections. A Bayesian inference tree shows the clustering of the 5′ and 3′ LTRs from various HERV-K (HML-2) proviruses. The K111 5′ LTR (red) and the 3′ LTRs of K111 (blue) and K222 (gray) proviruses cluster in three independent clades with a common ancestor. Posterior probability values  >  70 are shown.

    Techniques Used: Recombinant, Isolation, Sequencing

    Mapping of K222 proviruses in the human genome.  (A)  Schematic representation of the primer sets used to isolate K222 by PCR. The genomic structure of a centromeric provirus K111 is shown; the viral genes  gag ,  pro ,  pol ,  env , and  np9 , surrounded by LTRs, integrated into centromeric repeats (CER:D22Z3). The target site duplication of K111 GAATTC is indicated. The primers P1 and P2 bind CER:D22Z3. These primers were used in combination with primers that span the provirus genome. Arrows indicate the position and orientation of the primers; the number above indicates the nucleotide position they bind in reference to K111. Mapping to the 5′ end of the provirus was performed using the primer P1 and a set of HERV-K (HML-2) reverse primers. Mapping to the 3′ end of the provirus was performed with the reverse primer P2 and a set of HERV-K (HML-2) forward primers.  (B, C)  Isolation of K222 provirus. The sequence of K222 was detected by PCR from DNA of the cell lines H9 and HUT78, which lack K111 5′ end. Normal human DNA, containing K111, was used as a control for the PCR reaction. The number shown for each lane represents the primers. The gels show the amplification products of the 5′ mapping  (B)  or 3′ mapping  (C)  of centromeric proviruses in H9, HUT78, and normal human DNA using different combinations of primers. A molecular size ladder is indicated at the left. No amplification products were detected in H9 and HUT78 cell lines, in contrast to normal human DNA, when using the primer sets P1-982R, P1-2499R  (B) , or primer sets P2-1965F, and P2-2641F  (C) . An asterisk indicates a band that was shown by sequencing to be the result of non-specific amplification. Sequencing of the mapping products obtained from DNA of H9 and HUT78 cells reveals the sequence of K222.
    Figure Legend Snippet: Mapping of K222 proviruses in the human genome. (A) Schematic representation of the primer sets used to isolate K222 by PCR. The genomic structure of a centromeric provirus K111 is shown; the viral genes gag , pro , pol , env , and np9 , surrounded by LTRs, integrated into centromeric repeats (CER:D22Z3). The target site duplication of K111 GAATTC is indicated. The primers P1 and P2 bind CER:D22Z3. These primers were used in combination with primers that span the provirus genome. Arrows indicate the position and orientation of the primers; the number above indicates the nucleotide position they bind in reference to K111. Mapping to the 5′ end of the provirus was performed using the primer P1 and a set of HERV-K (HML-2) reverse primers. Mapping to the 3′ end of the provirus was performed with the reverse primer P2 and a set of HERV-K (HML-2) forward primers. (B, C) Isolation of K222 provirus. The sequence of K222 was detected by PCR from DNA of the cell lines H9 and HUT78, which lack K111 5′ end. Normal human DNA, containing K111, was used as a control for the PCR reaction. The number shown for each lane represents the primers. The gels show the amplification products of the 5′ mapping (B) or 3′ mapping (C) of centromeric proviruses in H9, HUT78, and normal human DNA using different combinations of primers. A molecular size ladder is indicated at the left. No amplification products were detected in H9 and HUT78 cell lines, in contrast to normal human DNA, when using the primer sets P1-982R, P1-2499R (B) , or primer sets P2-1965F, and P2-2641F (C) . An asterisk indicates a band that was shown by sequencing to be the result of non-specific amplification. Sequencing of the mapping products obtained from DNA of H9 and HUT78 cells reveals the sequence of K222.

    Techniques Used: Polymerase Chain Reaction, Isolation, Sequencing, Amplification

    Detection of K222 and recombinant K222/K111 sequences in individuals lacking the K111 5′ end.  (A)  Amplification of K222/K111 recombinant sequences. K222/K111 sequences were amplified with the primer 7972F and the primer P2, which binds to the K111 3′ flanking sequence (see Figure   2 ) in the DNA from individuals who lack the K111 5′ end (68, 90, and 95) and the cell line HUT78, which also lacks the K111 integration. As a positive control we used the DNA of individual 96, who is positive for K111 5′ end.  (B)  Amplification of K222 3′ integration. K222 was amplified with the primer 7972F and K222LTR-pCER:D22Z8R, the latter primer binding to the LTR-pCER:D22Z8 junction sequence present in K222, but not in K111. K111 3′ integration instead has a 5 bp sequence from the LTR and the target site duplication GAATTC not present in K222. Amplification of K222 3′ integration was seen in individuals having (96) or lacking (68, 90, and HUT78) the K111 5′ end.  (C)  Evolution of K222 and K222/K111 recombinant sequences in humans. A Bayesian inference tree of K222 and K222/K111 LTR sequences obtained by PCR in individuals lacking the K111 5′ end. The K222 sequences amplified are indicated with a K222 label. The tree reveals two different K222 LTR clades; K222 sequences similar to the K222 provirus (blue) and sequences that cluster to the K111 provirus (red). K222 sequences in individuals lacking the K111 5′ end clustering to K111 indicate the likely existence of K111 in the ancestral human lineage of those individuals. The K222/K111 recombinant clade (red) also suggests that K222 and K111 likely recombined by recombination/gene conversion during human evolution before K111 was lost from the lineage. Posterior probability values  > 85 are shown for the best tree.
    Figure Legend Snippet: Detection of K222 and recombinant K222/K111 sequences in individuals lacking the K111 5′ end. (A) Amplification of K222/K111 recombinant sequences. K222/K111 sequences were amplified with the primer 7972F and the primer P2, which binds to the K111 3′ flanking sequence (see Figure  2 ) in the DNA from individuals who lack the K111 5′ end (68, 90, and 95) and the cell line HUT78, which also lacks the K111 integration. As a positive control we used the DNA of individual 96, who is positive for K111 5′ end. (B) Amplification of K222 3′ integration. K222 was amplified with the primer 7972F and K222LTR-pCER:D22Z8R, the latter primer binding to the LTR-pCER:D22Z8 junction sequence present in K222, but not in K111. K111 3′ integration instead has a 5 bp sequence from the LTR and the target site duplication GAATTC not present in K222. Amplification of K222 3′ integration was seen in individuals having (96) or lacking (68, 90, and HUT78) the K111 5′ end. (C) Evolution of K222 and K222/K111 recombinant sequences in humans. A Bayesian inference tree of K222 and K222/K111 LTR sequences obtained by PCR in individuals lacking the K111 5′ end. The K222 sequences amplified are indicated with a K222 label. The tree reveals two different K222 LTR clades; K222 sequences similar to the K222 provirus (blue) and sequences that cluster to the K111 provirus (red). K222 sequences in individuals lacking the K111 5′ end clustering to K111 indicate the likely existence of K111 in the ancestral human lineage of those individuals. The K222/K111 recombinant clade (red) also suggests that K222 and K111 likely recombined by recombination/gene conversion during human evolution before K111 was lost from the lineage. Posterior probability values > 85 are shown for the best tree.

    Techniques Used: Recombinant, Amplification, Sequencing, Positive Control, Binding Assay, Polymerase Chain Reaction

    Absence of K111 5′ end in the genome of some cell lines.  (A)  Genomic structure of the K111 provirus. Arrows indicate the position of the primers P1 and P4, which amplify the 5′ integration of K111, and the primer/probe combination K111F, K111R, and K111P that specifically discriminates the K111 and K222  env  gene from other HERV-K (HML-2)  env  sequences due to a 6 bp mutation [  10 ].  (B)  Detection of K111 5′ end insertions in human cell lines. The 5′ flanking K111 insertions were detected in all human cell lines tested in this study by PCR using the primers P1 and P4 [  10 ], except for the DNA of cell lines H9, HUT78, H9/HTLVIII, and the IRA B-cell line. Arrows indicate individual K111 insertional polymorphisms. Integrity of the DNA was assessed by amplification of GAPDH (see lower gel). The molecular size of the DNA ladder is shown on the left of the gel. On top of each lane is the name of each cell line subjected to study. The weak bands observed in H9 and H9/HTLVIII were shown by sequencing to be the result of non-specific PCR amplification.
    Figure Legend Snippet: Absence of K111 5′ end in the genome of some cell lines. (A) Genomic structure of the K111 provirus. Arrows indicate the position of the primers P1 and P4, which amplify the 5′ integration of K111, and the primer/probe combination K111F, K111R, and K111P that specifically discriminates the K111 and K222 env gene from other HERV-K (HML-2) env sequences due to a 6 bp mutation [ 10 ]. (B) Detection of K111 5′ end insertions in human cell lines. The 5′ flanking K111 insertions were detected in all human cell lines tested in this study by PCR using the primers P1 and P4 [ 10 ], except for the DNA of cell lines H9, HUT78, H9/HTLVIII, and the IRA B-cell line. Arrows indicate individual K111 insertional polymorphisms. Integrity of the DNA was assessed by amplification of GAPDH (see lower gel). The molecular size of the DNA ladder is shown on the left of the gel. On top of each lane is the name of each cell line subjected to study. The weak bands observed in H9 and H9/HTLVIII were shown by sequencing to be the result of non-specific PCR amplification.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Sequencing

    Detection of K111 and K222 in the human population.  (A)  Genomic organization of K111 and K222 proviruses. The location of the primers to map K111 and K222 is shown.  (B)  Detection of K111 5′ end in the human population. The 5′ end of K111 was detected using the primers P1 and P4. The black arrow A indicates the K111 5′ end. The gray arrow indicates non-specific PCR products. On top of each lane is a number signifying each individual subjected to study.  (C, D)  Mapping of K111  (C)  and K222  (D)  in five individuals, who are positive or negative for the K111 5′ end, respectively. K111 mapping  (C)  was carried out with primer P1 and reverse primers that bind at positions 982, 2499, and 3460 bp of a K111 provirus. Black arrows indicate specific K111 insertions; A (product P1-982R), C (product P1-2499R), and D (product P1-3460R). The gray arrow indicates non-specific PCR amplifications. K111 detection was observed in the individuals labeled with the numbers, 1, 2, 3, 5, and 6, which are positive for the 5′ K111 end. Non-specific PCR product was detected in individuals labeled with the numbers 4, 68, 86, 90, and 95, which are negative for the 5′ K111 end as shown in B. The primers P1 and 3460R also detect K222 in individuals either negative or positive for the 5′ K111 integration (see stars). K222 mapping was carried out with the primer K222F and reverse primers that bind at positions 982, 1968, 2499, and 3460 bp in reference to K111. PCR products A, B, and C (black arrows) seen in the DNA of K111 positive individuals were shown to be the amplification of K111. No amplification products were seen in individuals lacking the 5′ end of K111. D represents the amplification product of K222.
    Figure Legend Snippet: Detection of K111 and K222 in the human population. (A) Genomic organization of K111 and K222 proviruses. The location of the primers to map K111 and K222 is shown. (B) Detection of K111 5′ end in the human population. The 5′ end of K111 was detected using the primers P1 and P4. The black arrow A indicates the K111 5′ end. The gray arrow indicates non-specific PCR products. On top of each lane is a number signifying each individual subjected to study. (C, D) Mapping of K111 (C) and K222 (D) in five individuals, who are positive or negative for the K111 5′ end, respectively. K111 mapping (C) was carried out with primer P1 and reverse primers that bind at positions 982, 2499, and 3460 bp of a K111 provirus. Black arrows indicate specific K111 insertions; A (product P1-982R), C (product P1-2499R), and D (product P1-3460R). The gray arrow indicates non-specific PCR amplifications. K111 detection was observed in the individuals labeled with the numbers, 1, 2, 3, 5, and 6, which are positive for the 5′ K111 end. Non-specific PCR product was detected in individuals labeled with the numbers 4, 68, 86, 90, and 95, which are negative for the 5′ K111 end as shown in B. The primers P1 and 3460R also detect K222 in individuals either negative or positive for the 5′ K111 integration (see stars). K222 mapping was carried out with the primer K222F and reverse primers that bind at positions 982, 1968, 2499, and 3460 bp in reference to K111. PCR products A, B, and C (black arrows) seen in the DNA of K111 positive individuals were shown to be the amplification of K111. No amplification products were seen in individuals lacking the 5′ end of K111. D represents the amplification product of K222.

    Techniques Used: Polymerase Chain Reaction, Labeling, Amplification

    14) Product Images from "Detection of large deletions in the LDL receptor gene with quantitative PCR methods"

    Article Title: Detection of large deletions in the LDL receptor gene with quantitative PCR methods

    Journal: BMC Medical Genetics

    doi: 10.1186/1471-2350-6-15

    Relative copy number of LDL receptor gene exon 5 . Boxplots of relative copy number of LDL receptor gene exon 5 measured with Real-Time PCR Analysis and MLPA analysis showing median; box: 25 th -75 th percentile; bars: largest and smallest values within 1.5 box lengths; circles: outliers.
    Figure Legend Snippet: Relative copy number of LDL receptor gene exon 5 . Boxplots of relative copy number of LDL receptor gene exon 5 measured with Real-Time PCR Analysis and MLPA analysis showing median; box: 25 th -75 th percentile; bars: largest and smallest values within 1.5 box lengths; circles: outliers.

    Techniques Used: Real-time Polymerase Chain Reaction, Multiplex Ligation-dependent Probe Amplification

    Relative copy number of LDL receptor gene exon 5 . Boxplots of relative copy number of LDL receptor gene exon 5 measured with Real-Time PCR Analysis and MLPA analysis showing median; box: 25 th -75 th percentile; bars: largest and smallest values within 1.5 box lengths; circles: outliers.
    Figure Legend Snippet: Relative copy number of LDL receptor gene exon 5 . Boxplots of relative copy number of LDL receptor gene exon 5 measured with Real-Time PCR Analysis and MLPA analysis showing median; box: 25 th -75 th percentile; bars: largest and smallest values within 1.5 box lengths; circles: outliers.

    Techniques Used: Real-time Polymerase Chain Reaction, Multiplex Ligation-dependent Probe Amplification

    15) Product Images from "Extensive somatic L1 retrotransposition in colorectal tumors"

    Article Title: Extensive somatic L1 retrotransposition in colorectal tumors

    Journal: Genome Research

    doi: 10.1101/gr.145235.112

    Genomic distribution of L1 insertions. Outer rings show the density of detected insertion sites for reference (gray) and nonreference (black) L1s. The approximate locations of the 72 PCR-validated somatic insertions are indicated by dots inside the circle.
    Figure Legend Snippet: Genomic distribution of L1 insertions. Outer rings show the density of detected insertion sites for reference (gray) and nonreference (black) L1s. The approximate locations of the 72 PCR-validated somatic insertions are indicated by dots inside the circle.

    Techniques Used: Polymerase Chain Reaction

    Analysis of factors influencing L1 activity. ( A ) L1 CpG promoter methylation status performed by quantitative bisulfite PCR analysis. (N) Normal tissue; (T) tumor tissue; (*) MSI. Replicates of four were done for each data point. (Error bars) Standard
    Figure Legend Snippet: Analysis of factors influencing L1 activity. ( A ) L1 CpG promoter methylation status performed by quantitative bisulfite PCR analysis. (N) Normal tissue; (T) tumor tissue; (*) MSI. Replicates of four were done for each data point. (Error bars) Standard

    Techniques Used: Activity Assay, Methylation, Polymerase Chain Reaction

    PCR validation scheme of L1-seq results. ( A ) The three-step PCR validation scheme and location of primers used. Triangles symbolize TSD. ( B ) PCR validation of the 3′ junction (ins. 7). This insertion is in tumor 1 of the eight DNA samples that
    Figure Legend Snippet: PCR validation scheme of L1-seq results. ( A ) The three-step PCR validation scheme and location of primers used. Triangles symbolize TSD. ( B ) PCR validation of the 3′ junction (ins. 7). This insertion is in tumor 1 of the eight DNA samples that

    Techniques Used: Polymerase Chain Reaction

    16) Product Images from "Expression of a large LINE-1-driven antisense RNA is linked to epigenetic silencing of the metastasis suppressor gene TFPI-2 in cancer"

    Article Title: Expression of a large LINE-1-driven antisense RNA is linked to epigenetic silencing of the metastasis suppressor gene TFPI-2 in cancer

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt438

    LCT13 and TFPI-2as expression is linked. ( A ) Schematic diagram of the genomic region in Figure 1 A indicating regions (1–7) analysed by strand-specific RT–PCR (middle). Shown above and below the schematic are the ethidium bromide–stained gels used to visualize the strand-specific RT–PCR. Regions 2–7 are specifically expressed in cancer cell lines (H, HCC-1954 and M, MCF-7), but not normal breast (N), showing that cancer-specific antisense transcription is detectable up to 300 kb away from the TFPI-2 gene and up to the LINE-1 retrotransposon associated with LCT13. ( B ) siRNA knockdown of the LCT13 transcript. 2D densitometry of semiquantitative strand-specific RT–PCR analysis normalized to APRT control reveals an approximate 50% knockdown in LCT13 levels in cells transfected with a pool of three siRNA duplexes directed against LCT13 compared to those transfected with scrambled control siRNAs (left panel). This is paralleled by a 40–50% decrease in the TFPI-2as transcript (right panel).
    Figure Legend Snippet: LCT13 and TFPI-2as expression is linked. ( A ) Schematic diagram of the genomic region in Figure 1 A indicating regions (1–7) analysed by strand-specific RT–PCR (middle). Shown above and below the schematic are the ethidium bromide–stained gels used to visualize the strand-specific RT–PCR. Regions 2–7 are specifically expressed in cancer cell lines (H, HCC-1954 and M, MCF-7), but not normal breast (N), showing that cancer-specific antisense transcription is detectable up to 300 kb away from the TFPI-2 gene and up to the LINE-1 retrotransposon associated with LCT13. ( B ) siRNA knockdown of the LCT13 transcript. 2D densitometry of semiquantitative strand-specific RT–PCR analysis normalized to APRT control reveals an approximate 50% knockdown in LCT13 levels in cells transfected with a pool of three siRNA duplexes directed against LCT13 compared to those transfected with scrambled control siRNAs (left panel). This is paralleled by a 40–50% decrease in the TFPI-2as transcript (right panel).

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Transfection

    A human TFPI-2 transgene is sensitive to antisense RNA repression in mouse ES cells. (A) Schematic diagram of constructs introduced into mouse ES cells: pTFPI-2as is designed to transcribe antisense to TFPI-2 from a CMV promoter, while pTFPI-2pa has a poly-A signal insertion downstream of the CMV promoter to block antisense transcription. Arrows indicate direction of transcription. Regions analysed by ChIP are annotated as ‘prom’ and ‘ex-in2’. ( B ) Strand-specific RT–PCR analysis of TFPI-2 antisenese (TFPI-2as) expression in transgenic mouse ES cell lines demonstrates increased levels in pTFPI-2as lines (L2 and L12) relative to pTFPI-2pa cells (L7 and L9), mouse Aprt acts as a positive control for RNA quality and quantity. This correlates with a reduction in TFPI-2 expression as shown by real-time PCR normalized to mouse Gapdh . ( C ) ChIP analysis followed by real-time PCR. Left panel: Antibodies to H3K9me3 reveal localized enrichment of H3K9me3 in the promoter region in the antisense expressing cell line, pTFPI-2as (L2), compared to cells transfected with pTFPI-2pa (L9), which express low levels of TFPI-2as. Right panel: Antibodies to H4K20me3 also show enrichment at the TFPI-2 promoter in pTFPI-2as compared to pTFPI-2pa.
    Figure Legend Snippet: A human TFPI-2 transgene is sensitive to antisense RNA repression in mouse ES cells. (A) Schematic diagram of constructs introduced into mouse ES cells: pTFPI-2as is designed to transcribe antisense to TFPI-2 from a CMV promoter, while pTFPI-2pa has a poly-A signal insertion downstream of the CMV promoter to block antisense transcription. Arrows indicate direction of transcription. Regions analysed by ChIP are annotated as ‘prom’ and ‘ex-in2’. ( B ) Strand-specific RT–PCR analysis of TFPI-2 antisenese (TFPI-2as) expression in transgenic mouse ES cell lines demonstrates increased levels in pTFPI-2as lines (L2 and L12) relative to pTFPI-2pa cells (L7 and L9), mouse Aprt acts as a positive control for RNA quality and quantity. This correlates with a reduction in TFPI-2 expression as shown by real-time PCR normalized to mouse Gapdh . ( C ) ChIP analysis followed by real-time PCR. Left panel: Antibodies to H3K9me3 reveal localized enrichment of H3K9me3 in the promoter region in the antisense expressing cell line, pTFPI-2as (L2), compared to cells transfected with pTFPI-2pa (L9), which express low levels of TFPI-2as. Right panel: Antibodies to H4K20me3 also show enrichment at the TFPI-2 promoter in pTFPI-2as compared to pTFPI-2pa.

    Techniques Used: Construct, Blocking Assay, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Expressing, Transgenic Assay, Positive Control, Real-time Polymerase Chain Reaction, Transfection

    Correlated expression of LCT13 and TFPI-2as transcripts in breast cancer cells. ( A ) Schematic diagram of a 300-kb region of chromosome 7q21.3 including LCT13 and the TFPI-2 gene. Scale is kilobase and indicates the position from the centromere with the value of 0 arbitrarily assigned to the TSS of CALCR . Genes (5′ segment of CALCR , TFPI-2 and GNGT1 ) are indicated as gray arrows. Two LINE-1 elements are present in the region (L1PA2 and L1PA6). Transcriptional orientations are indicated by arrows. LCT13 is a previously identified transcript shown to initiate at an L1ASP by 5′ RACE ( 22 ). TFPI-2as is the fragment analysed by strand-specific RT–PCR to test for the presence of TFPI-2 antisense RNAs. Displayed are the three spliced ESTs isolated from kidney (BG432114) and liver (DW466562 and DW435092) libraries that initiate at the LINE1 antisense promoter like LCT13 and extend past the TFPI-2 gene with a putative alternative transcript GNGT1-005 also annotated. ( B ) Expression of TFPI-2as (upper) and TFPI-2 (lower) in normal breast (N) and in breast cancer cell lines (H, HCC-1954; M, MCF7) analysed by strand specific and real-time RT–PCR, respectively. TFPI-2 expression is reduced in both breast cancer cell lines compared to normal controls (n = 3). TFPI-2 expression levels were normalized to HPRT . ( C ) Expression of TFPI-2as (upper) and TFPI-2 (lower) in a panel of five matched normal and tumour breast tissue analysed as described in B.
    Figure Legend Snippet: Correlated expression of LCT13 and TFPI-2as transcripts in breast cancer cells. ( A ) Schematic diagram of a 300-kb region of chromosome 7q21.3 including LCT13 and the TFPI-2 gene. Scale is kilobase and indicates the position from the centromere with the value of 0 arbitrarily assigned to the TSS of CALCR . Genes (5′ segment of CALCR , TFPI-2 and GNGT1 ) are indicated as gray arrows. Two LINE-1 elements are present in the region (L1PA2 and L1PA6). Transcriptional orientations are indicated by arrows. LCT13 is a previously identified transcript shown to initiate at an L1ASP by 5′ RACE ( 22 ). TFPI-2as is the fragment analysed by strand-specific RT–PCR to test for the presence of TFPI-2 antisense RNAs. Displayed are the three spliced ESTs isolated from kidney (BG432114) and liver (DW466562 and DW435092) libraries that initiate at the LINE1 antisense promoter like LCT13 and extend past the TFPI-2 gene with a putative alternative transcript GNGT1-005 also annotated. ( B ) Expression of TFPI-2as (upper) and TFPI-2 (lower) in normal breast (N) and in breast cancer cell lines (H, HCC-1954; M, MCF7) analysed by strand specific and real-time RT–PCR, respectively. TFPI-2 expression is reduced in both breast cancer cell lines compared to normal controls (n = 3). TFPI-2 expression levels were normalized to HPRT . ( C ) Expression of TFPI-2as (upper) and TFPI-2 (lower) in a panel of five matched normal and tumour breast tissue analysed as described in B.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Quantitative RT-PCR

    17) Product Images from "A Founder Large Deletion Mutation in Xeroderma Pigmentosum-Variant Form in Tunisia: Implication for Molecular Diagnosis and Therapy"

    Article Title: A Founder Large Deletion Mutation in Xeroderma Pigmentosum-Variant Form in Tunisia: Implication for Molecular Diagnosis and Therapy

    Journal: BioMed Research International

    doi: 10.1155/2014/256245

    Agar gel electrophoretic analysis of the PCR POLH gDNA of exon 10 and its intronic boundaries showed difference in the size between affected individuals (XPV17B-1 and XPV91) compared to healthy parents (XPV(P)) and a healthy control. (Marker: 1 kb DNA ladder molecular size marker (GeneRuler).)
    Figure Legend Snippet: Agar gel electrophoretic analysis of the PCR POLH gDNA of exon 10 and its intronic boundaries showed difference in the size between affected individuals (XPV17B-1 and XPV91) compared to healthy parents (XPV(P)) and a healthy control. (Marker: 1 kb DNA ladder molecular size marker (GeneRuler).)

    Techniques Used: Polymerase Chain Reaction, Marker

    18) Product Images from "High occurrence of BRCA1 intragenic rearrangements in hereditary breast and ovarian cancer syndrome in the Czech Republic"

    Article Title: High occurrence of BRCA1 intragenic rearrangements in hereditary breast and ovarian cancer syndrome in the Czech Republic

    Journal: BMC Medical Genetics

    doi: 10.1186/1471-2350-8-32

    Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 1A/1B-2 by long-range PCR and sequencing of the breakpoints. (B) Confirmation of the deletion of exons 5–14 by long-range PCR and sequencing of the breakpoints. (C) Confirmation of the deletion of exons 11–12 by long-range PCR and sequencing of the breakpoints. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).
    Figure Legend Snippet: Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 1A/1B-2 by long-range PCR and sequencing of the breakpoints. (B) Confirmation of the deletion of exons 5–14 by long-range PCR and sequencing of the breakpoints. (C) Confirmation of the deletion of exons 11–12 by long-range PCR and sequencing of the breakpoints. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

    Techniques Used: Polymerase Chain Reaction, Sequencing, Negative Control, Marker

    Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 18–19 by long-range PCR and sequencing of the breakpoints. (B) Confirmation of the deletion of exon 20 and sequencing of the breakpoints.Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).
    Figure Legend Snippet: Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 18–19 by long-range PCR and sequencing of the breakpoints. (B) Confirmation of the deletion of exon 20 and sequencing of the breakpoints.Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

    Techniques Used: Polymerase Chain Reaction, Sequencing, Negative Control, Marker

    Confirmation and characterization of the rearrangements . Confirmation of the deletion of the exons 21–22 by long-range PCR and sequencing of the breakpoints. The deletion/insertion event was characterized as g.77128_80906del3779ins236. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).
    Figure Legend Snippet: Confirmation and characterization of the rearrangements . Confirmation of the deletion of the exons 21–22 by long-range PCR and sequencing of the breakpoints. The deletion/insertion event was characterized as g.77128_80906del3779ins236. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

    Techniques Used: Polymerase Chain Reaction, Sequencing, Negative Control, Marker

    19) Product Images from "The physiological level of rNMPs present in mtDNA does not compromise its stability"

    Article Title: The physiological level of rNMPs present in mtDNA does not compromise its stability

    Journal: bioRxiv

    doi: 10.1101/746719

    Deletion of SAMHD1 does not affect mtDNA stability. a) MtDNA copy number in the TA muscle of 5 or 6 wt (filled dots) and SAMHD1 −/− (open dots) 13-week-old (adult), 1-year-old (old adult), and 2-year-old (aged) animals was determined by qPCR and normalized to the value for adult wt mice. The mean for each group is indicated by a horizontal line. The p-values were calculated using Welch’s t-test; ns, non-significant. b) DNA isolated from embryos and from the TA muscle of pups, adults, 1-year-old (old) adults, or aged animals was linearized with SacI endonuclease and separated on a neutral gel. MtDNA was visualized as above. Full-length mtDNA is indicated (FL); the asterisk denotes a higher-migrating species resistant to cleavage. c) Long-range PCR to detect deletions in mtDNA from the TA muscle of wt mice of various ages. Full-length product is indicated (FL). Only minor species containing deletions are observed in the mtDNA from old adults and aged animals, as indicated by the vertical line on the right-hand side of the gel. d) Untreated or alkali-treated DNA from skeletal muscle of aged wt and SAMHD1 −/− (ko) mice was analyzed on a denaturing gel, and mtDNA was visualized using a COX1 probe. Each sample lane corresponds to an individual mouse, and dotted lines represent the median. e) The median length of the untreated mtDNA in samples from Fig. 5d is indicated by a horizontal line. The two groups were compared using Welch’s t-test (ns, non-significant; n = 4). f) The length difference between untreated and alkali-treated mtDNAs shown in Fig. 5d was used to compute the number of rNMPs per single strand of mtDNA. The horizontal lines indicate the median. The p-value of the statistically significant difference between the two groups was calculated by Welch’s t-test; n = 4. g) Long-range PCR was performed on mtDNA isolated from the TA muscle of adult and aged wt or SAMHD1 −/− (ko) mice. FL, full-length product; the vertical line indicates the size range of mtDNA molecules with deletions. h) Kaplan–Meier survival curve for wt and SAMHD1 −/− (ko) mice. Comparison of the curves by the log-rank (Mantel–Cox) test confirmed no statistically significant difference between the genotypes. The sizes of the bands in the DNA ladder are indicated in kb. See also Fig. S5.
    Figure Legend Snippet: Deletion of SAMHD1 does not affect mtDNA stability. a) MtDNA copy number in the TA muscle of 5 or 6 wt (filled dots) and SAMHD1 −/− (open dots) 13-week-old (adult), 1-year-old (old adult), and 2-year-old (aged) animals was determined by qPCR and normalized to the value for adult wt mice. The mean for each group is indicated by a horizontal line. The p-values were calculated using Welch’s t-test; ns, non-significant. b) DNA isolated from embryos and from the TA muscle of pups, adults, 1-year-old (old) adults, or aged animals was linearized with SacI endonuclease and separated on a neutral gel. MtDNA was visualized as above. Full-length mtDNA is indicated (FL); the asterisk denotes a higher-migrating species resistant to cleavage. c) Long-range PCR to detect deletions in mtDNA from the TA muscle of wt mice of various ages. Full-length product is indicated (FL). Only minor species containing deletions are observed in the mtDNA from old adults and aged animals, as indicated by the vertical line on the right-hand side of the gel. d) Untreated or alkali-treated DNA from skeletal muscle of aged wt and SAMHD1 −/− (ko) mice was analyzed on a denaturing gel, and mtDNA was visualized using a COX1 probe. Each sample lane corresponds to an individual mouse, and dotted lines represent the median. e) The median length of the untreated mtDNA in samples from Fig. 5d is indicated by a horizontal line. The two groups were compared using Welch’s t-test (ns, non-significant; n = 4). f) The length difference between untreated and alkali-treated mtDNAs shown in Fig. 5d was used to compute the number of rNMPs per single strand of mtDNA. The horizontal lines indicate the median. The p-value of the statistically significant difference between the two groups was calculated by Welch’s t-test; n = 4. g) Long-range PCR was performed on mtDNA isolated from the TA muscle of adult and aged wt or SAMHD1 −/− (ko) mice. FL, full-length product; the vertical line indicates the size range of mtDNA molecules with deletions. h) Kaplan–Meier survival curve for wt and SAMHD1 −/− (ko) mice. Comparison of the curves by the log-rank (Mantel–Cox) test confirmed no statistically significant difference between the genotypes. The sizes of the bands in the DNA ladder are indicated in kb. See also Fig. S5.

    Techniques Used: Real-time Polymerase Chain Reaction, Mouse Assay, Isolation, Polymerase Chain Reaction

    20) Product Images from "Next generation sequencing and comparative analyses of Xenopus mitogenomes"

    Article Title: Next generation sequencing and comparative analyses of Xenopus mitogenomes

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-13-496

    Ratios of nonsynonymous/synonymous (dN/dS) nucleotide substitutions between the protein-coding genes of xenopus mitochondrial genomes. Although the ratios differ considerably between genes, complexes and pairs of species, in all cases genes are evolving under negative (purifying) selective pressure (dN/dS
    Figure Legend Snippet: Ratios of nonsynonymous/synonymous (dN/dS) nucleotide substitutions between the protein-coding genes of xenopus mitochondrial genomes. Although the ratios differ considerably between genes, complexes and pairs of species, in all cases genes are evolving under negative (purifying) selective pressure (dN/dS

    Techniques Used:

    Xenopus borealis mitochondrial genome. The complete mitochondrial genome of Xenopus borealis (17,474 bp, drawn to scale) All 13 protein coding genes are shown as open arrows, 2 ribosomal RNAs as shaded arrows and 22 tRNAs as arrowed lines. Each tRNA is shown by its single letter amino acid code. The two leucine and two serine tRNAs are differentiated by their respective anti-codons. The direction of transcription is indicated by the arrows. Also shown is the non-coding D-loop (control region, black) and the position of the primers (LongF1/R2 and LongF2/R1) used to generate the two long-PCR amplicons, which were pooled and sequenced using 454 technology.
    Figure Legend Snippet: Xenopus borealis mitochondrial genome. The complete mitochondrial genome of Xenopus borealis (17,474 bp, drawn to scale) All 13 protein coding genes are shown as open arrows, 2 ribosomal RNAs as shaded arrows and 22 tRNAs as arrowed lines. Each tRNA is shown by its single letter amino acid code. The two leucine and two serine tRNAs are differentiated by their respective anti-codons. The direction of transcription is indicated by the arrows. Also shown is the non-coding D-loop (control region, black) and the position of the primers (LongF1/R2 and LongF2/R1) used to generate the two long-PCR amplicons, which were pooled and sequenced using 454 technology.

    Techniques Used: Polymerase Chain Reaction

    Phylogenetic estimates of the interrelationship of four xenopus species and two relatives based on Bayesian analysis of amino acids from concatenated protein coding sequences. Nodal support is given by posterior probabilities; branch-length scale indicates number of substitutions per site.
    Figure Legend Snippet: Phylogenetic estimates of the interrelationship of four xenopus species and two relatives based on Bayesian analysis of amino acids from concatenated protein coding sequences. Nodal support is given by posterior probabilities; branch-length scale indicates number of substitutions per site.

    Techniques Used:

    Sliding window analysis of complete mitochondrial genome sequences of xenopus frogs. The coloured lines show the value of nucleotide divergence K(JC) (average number of nucleotide substitutions per site between species with Jukes and Canor correction) in a sliding window analysis of window size 300 bp with step size 10 for: all four xenopus (black), ST v XL (green), ST v XB (light blue), ST v XV (dark blue), XL v XB (orange), XB v XV (turquoise) and XL and XV (red). Gene boundaries and primers and regions commonly used in DNA barcoding amphibians are indicated.
    Figure Legend Snippet: Sliding window analysis of complete mitochondrial genome sequences of xenopus frogs. The coloured lines show the value of nucleotide divergence K(JC) (average number of nucleotide substitutions per site between species with Jukes and Canor correction) in a sliding window analysis of window size 300 bp with step size 10 for: all four xenopus (black), ST v XL (green), ST v XB (light blue), ST v XV (dark blue), XL v XB (orange), XB v XV (turquoise) and XL and XV (red). Gene boundaries and primers and regions commonly used in DNA barcoding amphibians are indicated.

    Techniques Used:

    Long PCR, COX1, 16S, primer region 1 and primer region 2 amplicons. Agarose gel electrophoresis of ( A ) Xenopus borealis (XB; lanes 1 and 2) and X. victorianus (XV; lanes 3 and 4) PCR fragments using Long F1/R2 (lanes 1 and 3) and Long F2/R1 primers (lanes 2 and 4). ( B ) XB (lanes 1 and 2) and XV (lane 3) PCR fragments using COX1 (lane 1) and 16SA-Lmod/H (lanes 2 and 3) primers. ( C ) XB (lanes 1-2 and 5-6) and XV (lanes 3-4 and 7-8) PCR fragments using AMP1F/R (lanes 1-4) and AMP2F/R (lanes 5-8) primers. M1 and M2 = 1kb and 100bp DNA ladders, respectively.
    Figure Legend Snippet: Long PCR, COX1, 16S, primer region 1 and primer region 2 amplicons. Agarose gel electrophoresis of ( A ) Xenopus borealis (XB; lanes 1 and 2) and X. victorianus (XV; lanes 3 and 4) PCR fragments using Long F1/R2 (lanes 1 and 3) and Long F2/R1 primers (lanes 2 and 4). ( B ) XB (lanes 1 and 2) and XV (lane 3) PCR fragments using COX1 (lane 1) and 16SA-Lmod/H (lanes 2 and 3) primers. ( C ) XB (lanes 1-2 and 5-6) and XV (lanes 3-4 and 7-8) PCR fragments using AMP1F/R (lanes 1-4) and AMP2F/R (lanes 5-8) primers. M1 and M2 = 1kb and 100bp DNA ladders, respectively.

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis

    21) Product Images from "A Long ncRNA Links Copy Number Variation to a Polycomb/Trithorax Epigenetic Switch in FSHD Muscular Dystrophy"

    Article Title: A Long ncRNA Links Copy Number Variation to a Polycomb/Trithorax Epigenetic Switch in FSHD Muscular Dystrophy

    Journal: Cell

    doi: 10.1016/j.cell.2012.03.035

    DBE-T Is Nuclear, Chromatin-Associated, and Localized to the FSHD Locus (A) Total RNA from AZA+TSA treated chr4/CHO cells was separated into cytoplasmic, nuclear-soluble, and chromatin-bound fractions. The relative abundance of DBE-T in the different fractions was measured by qRT-PCR. As control, Gapdh and Xist were analyzed. The error bars represent SEM. (B) Schematic representation of the location of the DBE-T and D4Z4 probes. (C) Following AZA+TSA treatment, chr4/CHO cells were analyzed by RNA/DNA FISH. A single Z stack acquired with an Olympus IX70 DeltaVision RT Deconvolution System microscope is shown. Signals colocalization was detected in 98.8% of the double positive cells (n = 80) deriving from 3 independent experiments. DBE-T is visualized in green, and the D4Z4 DNA is in red. DAPI is in blue. See also Figures S6 and S7 .
    Figure Legend Snippet: DBE-T Is Nuclear, Chromatin-Associated, and Localized to the FSHD Locus (A) Total RNA from AZA+TSA treated chr4/CHO cells was separated into cytoplasmic, nuclear-soluble, and chromatin-bound fractions. The relative abundance of DBE-T in the different fractions was measured by qRT-PCR. As control, Gapdh and Xist were analyzed. The error bars represent SEM. (B) Schematic representation of the location of the DBE-T and D4Z4 probes. (C) Following AZA+TSA treatment, chr4/CHO cells were analyzed by RNA/DNA FISH. A single Z stack acquired with an Olympus IX70 DeltaVision RT Deconvolution System microscope is shown. Signals colocalization was detected in 98.8% of the double positive cells (n = 80) deriving from 3 independent experiments. DBE-T is visualized in green, and the D4Z4 DNA is in red. DAPI is in blue. See also Figures S6 and S7 .

    Techniques Used: Quantitative RT-PCR, Fluorescence In Situ Hybridization, Microscopy

    DBE-T Is a Mature RNA, Related to Figure 4 (A) Human chr4/CHO and CHO cells were collected in the de-repressed state (AZA+TSA). A set of samples were treated with RNase A+T1. RNA FISH quantification of DBE-T positive nuclei was performed in all conditions and it was normalized to signals in human chr4/CHO cells treated with AZA+TSA. The error bars represent SD. (B) AZA+TSA human chr4/CHO cells were treated (Right panels) or untreated (Left panels) with Actinomycin D at 5 μg/ml for 15′. Hybridization to β-actin (Top panels) or DBE-T (Bottom panels) RNAs was performed. The arrows indicate dots of nuclear RNAs. See Statistical Test section for statistical analysis. The error bars represent SD. DBE-T and β-actin RNAs are in red. DAPI is in blue. The images correspond to a single Z stack acquired with an Olympus IX70 DeltaVision RT Deconvolution System microscope. (A and B) The asterisks indicate statistically significant differences. A Two-tailed, paired, t test was applied. For DBE-T : human chr4/CHO AZA+TSA n = 4; human chr4/CHO AZA+TSA + Actinomycin D n = 4, p = 0.0914; human Chr4/CHO AZA+TSA + RNase A+T1 n = 4, p = 0.0056; CHO AZA+TSA n = 4, p = 0.0006. For β-actin : human chr4/CHO AZA+TSA n = 4; human chr4/CHO AZA+TSA + Actinomycin D n = 4, p = 0.0012.
    Figure Legend Snippet: DBE-T Is a Mature RNA, Related to Figure 4 (A) Human chr4/CHO and CHO cells were collected in the de-repressed state (AZA+TSA). A set of samples were treated with RNase A+T1. RNA FISH quantification of DBE-T positive nuclei was performed in all conditions and it was normalized to signals in human chr4/CHO cells treated with AZA+TSA. The error bars represent SD. (B) AZA+TSA human chr4/CHO cells were treated (Right panels) or untreated (Left panels) with Actinomycin D at 5 μg/ml for 15′. Hybridization to β-actin (Top panels) or DBE-T (Bottom panels) RNAs was performed. The arrows indicate dots of nuclear RNAs. See Statistical Test section for statistical analysis. The error bars represent SD. DBE-T and β-actin RNAs are in red. DAPI is in blue. The images correspond to a single Z stack acquired with an Olympus IX70 DeltaVision RT Deconvolution System microscope. (A and B) The asterisks indicate statistically significant differences. A Two-tailed, paired, t test was applied. For DBE-T : human chr4/CHO AZA+TSA n = 4; human chr4/CHO AZA+TSA + Actinomycin D n = 4, p = 0.0914; human Chr4/CHO AZA+TSA + RNase A+T1 n = 4, p = 0.0056; CHO AZA+TSA n = 4, p = 0.0006. For β-actin : human chr4/CHO AZA+TSA n = 4; human chr4/CHO AZA+TSA + Actinomycin D n = 4, p = 0.0012.

    Techniques Used: Fluorescence In Situ Hybridization, Hybridization, Microscopy, Two Tailed Test

    DBE-T Is a Long ncRNA Whose Transcription Starts outside the D4Z4 Repeat Array and Encompasses the NDE Region, Related to Figure 4 (A) Schematic representation of the regions analyzed. The asterisk corresponds to the Transcriptional Start Site (TSS) mapped by 5′ RACE. The position indicated is referred to AF117653. The arrows represent the primers used in panel C to amplify a single NDE/DBE-T transcript. Lines and numbers positioned in the lower part of the scheme correspond to the overlapping regions amplified in panel D. (B) Northern blot assays performed on PolyA+ RNA extracted from human chr4/CHO cells untreated (Control) or treated with AZA+TSA (AZA+TSA). Hybridizations with probes mapping to NDE, DBE, DUX4 and Gapdh , as loading control, are shown. (C) RT-PCR to amplify a single transcript from NDE to DBE was performed on RNA extracted from CHO cells transfected with a construct carrying the entire AF117653 sequence (pGEM42, derived from an FSHD patient, containing the 4q35 region ranging from upstream p13E-11, two D4Z4 repeats and the distal region, Kowaljow et al., 2007 ) or with the empty vector as control. As positive control, the pGEM42 plasmid DNA was PCR amplified; RT- and no template reactions were performed as negative controls. (D) The NDE-DBE region was divided in eight overlapping regions (see panel A) that were amplified by RT-PCR in RNA samples extracted from human chr4/CHO cells untreated (Control) or treated with AZA+TSA (AZA+TSA). As loading control, Gapdh was amplified. (E) NDE transcription was evaluated in the repressed (control) and de-repressed (AZA+TSA) states by qRT-PCR. Results are shown as expression over β-actin . The error bars represent SEM. (F) The relative enrichment of the NDE transcript in the indicated subcellular fractions as measured by qRT-PCR is shown. The error bars represent SEM. (G) Analysis of NDE expression by qRT-PCR in control and FSHD primary muscle cells. Results are shown as expression over GAPDH . The error bars represent SEM. (H) Expression of 4q35 genes and Gapdh , as control, in human chr4/CHO cells knockdown for NDE transcript (G left) in the de-repressed state (AZA+TSA). Results are shown as expression over β-actin . The error bars represent SEM. (I) The NDE transcript is downregulated upon DBE-T knockdown and DBE-T is downregulated upon NDE transcript knockdown. Results are shown as expression over β-actin . The error bars represent SEM.
    Figure Legend Snippet: DBE-T Is a Long ncRNA Whose Transcription Starts outside the D4Z4 Repeat Array and Encompasses the NDE Region, Related to Figure 4 (A) Schematic representation of the regions analyzed. The asterisk corresponds to the Transcriptional Start Site (TSS) mapped by 5′ RACE. The position indicated is referred to AF117653. The arrows represent the primers used in panel C to amplify a single NDE/DBE-T transcript. Lines and numbers positioned in the lower part of the scheme correspond to the overlapping regions amplified in panel D. (B) Northern blot assays performed on PolyA+ RNA extracted from human chr4/CHO cells untreated (Control) or treated with AZA+TSA (AZA+TSA). Hybridizations with probes mapping to NDE, DBE, DUX4 and Gapdh , as loading control, are shown. (C) RT-PCR to amplify a single transcript from NDE to DBE was performed on RNA extracted from CHO cells transfected with a construct carrying the entire AF117653 sequence (pGEM42, derived from an FSHD patient, containing the 4q35 region ranging from upstream p13E-11, two D4Z4 repeats and the distal region, Kowaljow et al., 2007 ) or with the empty vector as control. As positive control, the pGEM42 plasmid DNA was PCR amplified; RT- and no template reactions were performed as negative controls. (D) The NDE-DBE region was divided in eight overlapping regions (see panel A) that were amplified by RT-PCR in RNA samples extracted from human chr4/CHO cells untreated (Control) or treated with AZA+TSA (AZA+TSA). As loading control, Gapdh was amplified. (E) NDE transcription was evaluated in the repressed (control) and de-repressed (AZA+TSA) states by qRT-PCR. Results are shown as expression over β-actin . The error bars represent SEM. (F) The relative enrichment of the NDE transcript in the indicated subcellular fractions as measured by qRT-PCR is shown. The error bars represent SEM. (G) Analysis of NDE expression by qRT-PCR in control and FSHD primary muscle cells. Results are shown as expression over GAPDH . The error bars represent SEM. (H) Expression of 4q35 genes and Gapdh , as control, in human chr4/CHO cells knockdown for NDE transcript (G left) in the de-repressed state (AZA+TSA). Results are shown as expression over β-actin . The error bars represent SEM. (I) The NDE transcript is downregulated upon DBE-T knockdown and DBE-T is downregulated upon NDE transcript knockdown. Results are shown as expression over β-actin . The error bars represent SEM.

    Techniques Used: Amplification, Northern Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Construct, Sequencing, Derivative Assay, Plasmid Preparation, Positive Control, Polymerase Chain Reaction, Quantitative RT-PCR, Expressing

    DBE-T Directly Binds the TrxG Protein Ash1L and Recruits It to the FSHD Locus (A) RNA immunoprecipitation (IP) following UV crosslinking for Ash1L or IgG on AZA+TSA treated chr4/CHO cells. DBE-T or, as control, pre-miR19A and U1 snRNA enrichments were measured by qRT-PCR. The error bars represent SEM. (B) In vitro RNA-GST pull-down assay showing the interaction between recombinant GST-fused Ash1L SET domain or GST and in vitro transcribed DBE-T . On the right, Coomassie staining of purified recombinant proteins. After RNA recovery, samples were analyzed by qRT-PCR. The error bars represent SEM. (C) Following AZA+TSA treatment, chr4/CHO cells stably expressing a nonsilencing control shRNA or sh DBE-T were analyzed by ChIP for Ash1L or IgG. Enrichment for NDE was analyzed by qPCR and displayed as enrichment relative to input. The error bars represent SEM. (D) Upon AZA+TSA treatment, control shRNA, and sh Ash1L cells were collected to analyze DBE-T expression by qRT-PCR. Results are expressed over β-actin . The error bars represent SEM. See also Figure S8 .
    Figure Legend Snippet: DBE-T Directly Binds the TrxG Protein Ash1L and Recruits It to the FSHD Locus (A) RNA immunoprecipitation (IP) following UV crosslinking for Ash1L or IgG on AZA+TSA treated chr4/CHO cells. DBE-T or, as control, pre-miR19A and U1 snRNA enrichments were measured by qRT-PCR. The error bars represent SEM. (B) In vitro RNA-GST pull-down assay showing the interaction between recombinant GST-fused Ash1L SET domain or GST and in vitro transcribed DBE-T . On the right, Coomassie staining of purified recombinant proteins. After RNA recovery, samples were analyzed by qRT-PCR. The error bars represent SEM. (C) Following AZA+TSA treatment, chr4/CHO cells stably expressing a nonsilencing control shRNA or sh DBE-T were analyzed by ChIP for Ash1L or IgG. Enrichment for NDE was analyzed by qPCR and displayed as enrichment relative to input. The error bars represent SEM. (D) Upon AZA+TSA treatment, control shRNA, and sh Ash1L cells were collected to analyze DBE-T expression by qRT-PCR. Results are expressed over β-actin . The error bars represent SEM. See also Figure S8 .

    Techniques Used: Immunoprecipitation, Quantitative RT-PCR, In Vitro, Pull Down Assay, Recombinant, Staining, Purification, Stable Transfection, Expressing, shRNA, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    22) Product Images from "Endosymbiotic Gene Transfer in Tertiary Plastid-Containing Dinoflagellates"

    Article Title: Endosymbiotic Gene Transfer in Tertiary Plastid-Containing Dinoflagellates

    Journal: Eukaryotic Cell

    doi: 10.1128/EC.00299-13

    (A) Total GC content and GC content at third codon positions (GC3) for the 17 genes with diatom affinity in dinotoms. The sequence names are abbreviated as follows: Db, D. baltica ; Kf, K. foliaceum . (B) Frequency distribution of the GC content for all transcripts from F. cylindrus , P. tricornutum , and T. pseudonana and all contigs from the D. baltica SL library. Gray arrows denote the genes with diatom affinity with low GC content; pink arrows denote the genes with diatom affinity with high GC content.
    Figure Legend Snippet: (A) Total GC content and GC content at third codon positions (GC3) for the 17 genes with diatom affinity in dinotoms. The sequence names are abbreviated as follows: Db, D. baltica ; Kf, K. foliaceum . (B) Frequency distribution of the GC content for all transcripts from F. cylindrus , P. tricornutum , and T. pseudonana and all contigs from the D. baltica SL library. Gray arrows denote the genes with diatom affinity with low GC content; pink arrows denote the genes with diatom affinity with high GC content.

    Techniques Used: Sequencing

    23) Product Images from "Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells"

    Article Title: Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20131436

    Cellular senescence in Usp3 Δ/Δ HSC compartment and BM. (A) Cytospins of BM cells from Usp3 Δ/Δ and WT mice were assayed for SA-β-galactosidase activity. The percentage of SA- β -Gal–positive cells was quantified by counting 100 cells on three separate fields ( n = 4 mice per genotype). Bar, 20 µm. (B and C) Cytospin preparations of sorted LSKs from BM of Usp3 Δ/Δ or WT mice were immunostained for HP1γ (B) and H3K9Me3 (C). Focal nuclear staining is visible in insets. Signal intensity per nucleus was quantified by ImageJ. n = 3 per genotype. A minimum of 1,000 nuclei/sample was evaluated. Data are mean ± SEM of one of two representative experiments. Bars: 75 µm; (inset) 10 µm. (D) Immunostaining for HP1γ and H3K9Me3 on BM sections from Usp3 Δ/Δ and WT mice. The percentage of positive cells was quantified in 3 fields on a minimum of 1,500 cells/field per sample. n = 7 per genotype. Bar, 20 µm. (E) Quantification of apoptotic (Annexin V positive and Propidium Iodide [PI] negative) freshly isolated hematopoietic subpopulations (mean ± SD) from WT or Usp3 Δ/Δ. n = 3 per genotype. One of two representative experiments is shown. (F) Representative images of WT and Usp3 Δ/Δ BM sections stained for apoptosis-indicating cleavage (cl.) of caspase 3. n = 6 mice per genotype. Bar, 500 µm. (G) Sorted LT-HSCs were plated after 8 d (first plating) or 11 d (second plating) in culture and monitored for growth. Kinetic measures the number of cells, recorded over time and plotted as phase contrast object confluence. n = 4 wells per data point. Mean ± SD of one of two representative experiments is shown. Representative images at time 0 and 96 h after plating are shown. Bar, 300 µm. (H) Immunostaining of in vitro expanded LT-HSCs for H3K9Me3. Signal quantification by ImageJ from two independent experiments is shown (mean ± SEM). n = 150 per genotype. Bar, 10 µm. (I) LT-HSCs cultures were assayed for SA-β-galactosidase activity after 3, 8, or 11 d (dd) in culture. A minimum of 350 (3dd), 2300 (8 dd), or 550 (11 dd) cells counted in 10 separate fields were evaluated. Bar, 20 µm. (J) LT-HSCs cultures were assayed for SA-β-galactosidase activity upon Tat-cMyc protein transduction. A minimum of 1,000 cells per genotype was evaluated in two replicate experiments. Bar, 20 µm. Mice were 32 wk old (A–D) or 40–44 wk old (E and F). G–J: LT-HSCs for in vitro expansion were isolated from 40–44-wk-old mice. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001; ns, not significant.
    Figure Legend Snippet: Cellular senescence in Usp3 Δ/Δ HSC compartment and BM. (A) Cytospins of BM cells from Usp3 Δ/Δ and WT mice were assayed for SA-β-galactosidase activity. The percentage of SA- β -Gal–positive cells was quantified by counting 100 cells on three separate fields ( n = 4 mice per genotype). Bar, 20 µm. (B and C) Cytospin preparations of sorted LSKs from BM of Usp3 Δ/Δ or WT mice were immunostained for HP1γ (B) and H3K9Me3 (C). Focal nuclear staining is visible in insets. Signal intensity per nucleus was quantified by ImageJ. n = 3 per genotype. A minimum of 1,000 nuclei/sample was evaluated. Data are mean ± SEM of one of two representative experiments. Bars: 75 µm; (inset) 10 µm. (D) Immunostaining for HP1γ and H3K9Me3 on BM sections from Usp3 Δ/Δ and WT mice. The percentage of positive cells was quantified in 3 fields on a minimum of 1,500 cells/field per sample. n = 7 per genotype. Bar, 20 µm. (E) Quantification of apoptotic (Annexin V positive and Propidium Iodide [PI] negative) freshly isolated hematopoietic subpopulations (mean ± SD) from WT or Usp3 Δ/Δ. n = 3 per genotype. One of two representative experiments is shown. (F) Representative images of WT and Usp3 Δ/Δ BM sections stained for apoptosis-indicating cleavage (cl.) of caspase 3. n = 6 mice per genotype. Bar, 500 µm. (G) Sorted LT-HSCs were plated after 8 d (first plating) or 11 d (second plating) in culture and monitored for growth. Kinetic measures the number of cells, recorded over time and plotted as phase contrast object confluence. n = 4 wells per data point. Mean ± SD of one of two representative experiments is shown. Representative images at time 0 and 96 h after plating are shown. Bar, 300 µm. (H) Immunostaining of in vitro expanded LT-HSCs for H3K9Me3. Signal quantification by ImageJ from two independent experiments is shown (mean ± SEM). n = 150 per genotype. Bar, 10 µm. (I) LT-HSCs cultures were assayed for SA-β-galactosidase activity after 3, 8, or 11 d (dd) in culture. A minimum of 350 (3dd), 2300 (8 dd), or 550 (11 dd) cells counted in 10 separate fields were evaluated. Bar, 20 µm. (J) LT-HSCs cultures were assayed for SA-β-galactosidase activity upon Tat-cMyc protein transduction. A minimum of 1,000 cells per genotype was evaluated in two replicate experiments. Bar, 20 µm. Mice were 32 wk old (A–D) or 40–44 wk old (E and F). G–J: LT-HSCs for in vitro expansion were isolated from 40–44-wk-old mice. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001; ns, not significant.

    Techniques Used: Mouse Assay, Activity Assay, Staining, Immunostaining, Isolation, In Vitro, Transduction

    USP3 protects HSCs from genotoxic stress in vivo and in vitro. (A–C) Age-matched (8 wk old) WT ( n = 12) and Usp3 Δ/Δ ( n = 13) mice were exposed to 7 Gy TBI and monitored for 28 d. (A) Kaplan Meier survival curve. P-value was determined by Log-rank test. (B) WBC counts of unirradiated or irradiated mice (WT, 28 d after TBI) and Usp3 Δ/Δ (at time of sacrifice due to illness after TBI). Results are mean ± SD from two independent experiments. (C) Representative images of hematoxylin-eosin (H E)–stained tissue sections of WT (28 d after TBI) and of Usp3 Δ/Δ (at the time of sacrifice due to illness after 7 Gy TBI) mice. Bars: (BM) 100 µm; (spleen) 50 µm; (small intestine) 50 µm; (heart) 500 µm; (inset) 50 µm. (D) CFU-C from BM cells of 8-wk-old WT or Usp3 Δ/Δ mice. Mice ( n = 3 per genotype) were left untreated or subjected to TBI (5Gy). 7 d after IR, BM cells were isolated and plated on methylcellulose with cytokines. Results are means ± SD. (E and F) Absolute numbers (2 femurs and 2 hips bones) of Lin − , LSKs, and HSCs in 44-wk-old mice that were left untreated (UN) or exposed to 5 Gy TBI (5Gy) and sacrificed after 24 h (E). UN, n = 11 per genotype; IR, WT, n = 3; Usp3 Δ/Δ, n = 4. (F) Increased cell death in Usp3 Δ/Δ LSKs, as determined by Annexin V staining in mice as in E. UN, n = 3 per genotype; IR, WT, n = 3; Usp3 Δ/Δ, n = 4. Results are means ± SEM. (G and H) Immunofluorescence of γH2AX and 53BP1 in WT or Usp3 Δ/Δ LT-HSCs in culture for 8–10 d. IRIFs were scored in LT-HSCs after mock treatment or at 30 min and 24 h after 2Gy of IR. The percentage of cells containing > 5 IRIFs are plotted ± SD. Representative images for 53BP1 staining are shown (H). A minimum of 50 cells/sample/experiment over two (γH2AX) or three (53BP1) independent experiments was evaluated. Bar, 10 µm. (I) Percentage of micronuclei in WT or Usp3 Δ/Δ LT-HSCs in culture for 8–10 d. Cells were irradiated with 2Gy and micronuclei scored at 24 h after IR. Results are means of three independent experiments ± SD on a minimum of 70 cells/genotype. Bar, 5 µm. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ P ≤ 0.001; ns, not significant.
    Figure Legend Snippet: USP3 protects HSCs from genotoxic stress in vivo and in vitro. (A–C) Age-matched (8 wk old) WT ( n = 12) and Usp3 Δ/Δ ( n = 13) mice were exposed to 7 Gy TBI and monitored for 28 d. (A) Kaplan Meier survival curve. P-value was determined by Log-rank test. (B) WBC counts of unirradiated or irradiated mice (WT, 28 d after TBI) and Usp3 Δ/Δ (at time of sacrifice due to illness after TBI). Results are mean ± SD from two independent experiments. (C) Representative images of hematoxylin-eosin (H E)–stained tissue sections of WT (28 d after TBI) and of Usp3 Δ/Δ (at the time of sacrifice due to illness after 7 Gy TBI) mice. Bars: (BM) 100 µm; (spleen) 50 µm; (small intestine) 50 µm; (heart) 500 µm; (inset) 50 µm. (D) CFU-C from BM cells of 8-wk-old WT or Usp3 Δ/Δ mice. Mice ( n = 3 per genotype) were left untreated or subjected to TBI (5Gy). 7 d after IR, BM cells were isolated and plated on methylcellulose with cytokines. Results are means ± SD. (E and F) Absolute numbers (2 femurs and 2 hips bones) of Lin − , LSKs, and HSCs in 44-wk-old mice that were left untreated (UN) or exposed to 5 Gy TBI (5Gy) and sacrificed after 24 h (E). UN, n = 11 per genotype; IR, WT, n = 3; Usp3 Δ/Δ, n = 4. (F) Increased cell death in Usp3 Δ/Δ LSKs, as determined by Annexin V staining in mice as in E. UN, n = 3 per genotype; IR, WT, n = 3; Usp3 Δ/Δ, n = 4. Results are means ± SEM. (G and H) Immunofluorescence of γH2AX and 53BP1 in WT or Usp3 Δ/Δ LT-HSCs in culture for 8–10 d. IRIFs were scored in LT-HSCs after mock treatment or at 30 min and 24 h after 2Gy of IR. The percentage of cells containing > 5 IRIFs are plotted ± SD. Representative images for 53BP1 staining are shown (H). A minimum of 50 cells/sample/experiment over two (γH2AX) or three (53BP1) independent experiments was evaluated. Bar, 10 µm. (I) Percentage of micronuclei in WT or Usp3 Δ/Δ LT-HSCs in culture for 8–10 d. Cells were irradiated with 2Gy and micronuclei scored at 24 h after IR. Results are means of three independent experiments ± SD on a minimum of 70 cells/genotype. Bar, 5 µm. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ P ≤ 0.001; ns, not significant.

    Techniques Used: In Vivo, In Vitro, Mouse Assay, Irradiation, Staining, Isolation, Immunofluorescence

    USP3 deletion leads to a genome-wide increase in mono-ubiquitinated H2A (uH2A) and H2B (uH2B) in mouse cells and tissues. (A) Immunostaining of WT and Usp3 Δ/Δ MEFs with anti-Ub (FK2) antibody (red) and DAPI (blue). The FK2 signal intensity per nucleus was quantified by ImageJ. A minimum of 1,000 cells/sample was analyzed. Data are means ± SEM of two independent MEF lines per genotype. Bars: 500 µm; (inset) 10 µm. (B) WT or Usp3 Δ/Δ MEFs were infected with control retrovirus (empty vector, ev) or with retrovirus expressing WT USP3 (WT-USP3) and immunostained with FK2. Representative images and FK2 signal quantification as in A. Right panel: immunoblot of MEFs WCE for USP3 and CDK4 (*, nonspecific protein band). Data are means ± SEM of two independent experiments with a minimum of 800 cells/genotype. Bar, 500 µm. (C) Immunoblot of core histone fraction from WT and Usp3 Δ/Δ MEFs. Quantification by ImageJ of the uH2A and uH2B signal normalized, respectively, to H2A or H2B, averaged from four (uH2A) or three (uH2B) independent MEF lines per genotype is shown. Data are means ± SD. (D) FK2 staining on freshly isolated BM cells from WT and Usp3 Δ/Δ mice ( n = 3 per genotype). Signal intensity was quantified as in A. WT, n = 555; Usp3 Δ/Δ, n = 938. Data are means ± SEM. (E) Immunoblot of core histones fraction from liver and spleen of WT and Usp3 Δ/Δ. uH2A and uH2B were quantified as in C. uH2A, n = 3 mice; uH2B, n = 2 mice per genotype. Data are means ± SD. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
    Figure Legend Snippet: USP3 deletion leads to a genome-wide increase in mono-ubiquitinated H2A (uH2A) and H2B (uH2B) in mouse cells and tissues. (A) Immunostaining of WT and Usp3 Δ/Δ MEFs with anti-Ub (FK2) antibody (red) and DAPI (blue). The FK2 signal intensity per nucleus was quantified by ImageJ. A minimum of 1,000 cells/sample was analyzed. Data are means ± SEM of two independent MEF lines per genotype. Bars: 500 µm; (inset) 10 µm. (B) WT or Usp3 Δ/Δ MEFs were infected with control retrovirus (empty vector, ev) or with retrovirus expressing WT USP3 (WT-USP3) and immunostained with FK2. Representative images and FK2 signal quantification as in A. Right panel: immunoblot of MEFs WCE for USP3 and CDK4 (*, nonspecific protein band). Data are means ± SEM of two independent experiments with a minimum of 800 cells/genotype. Bar, 500 µm. (C) Immunoblot of core histone fraction from WT and Usp3 Δ/Δ MEFs. Quantification by ImageJ of the uH2A and uH2B signal normalized, respectively, to H2A or H2B, averaged from four (uH2A) or three (uH2B) independent MEF lines per genotype is shown. Data are means ± SD. (D) FK2 staining on freshly isolated BM cells from WT and Usp3 Δ/Δ mice ( n = 3 per genotype). Signal intensity was quantified as in A. WT, n = 555; Usp3 Δ/Δ, n = 938. Data are means ± SEM. (E) Immunoblot of core histones fraction from liver and spleen of WT and Usp3 Δ/Δ. uH2A and uH2B were quantified as in C. uH2A, n = 3 mice; uH2B, n = 2 mice per genotype. Data are means ± SD. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

    Techniques Used: Genome Wide, Immunostaining, Infection, Plasmid Preparation, Expressing, Staining, Isolation, Mouse Assay

    USP3-deficient HSCs accumulate spontaneous DNA damage. (A–C) γH2AX immunostaining on sorted Lin − Sca1 + c-Kit + CD150 + flk2/CD135 − CD34 − (LT-HSC) or CD34 + (ST-HSC) from 44-wk-old (A and B) or 17-wk-old (C) mice. Representative images (A) of LT-HSCs and ST-HSCs from 44-wk-old mice and quantification of the number of γH2AX foci/cell in HSCs from 44-wk-old (B) or 17-wk-old mice (minimum of 200 cells per genotype; C). Results are from two independent experiments. n = 3 mice/genotype/experiment. Bar, 5 µm. (D–F) Alkaline comet assay on sorted Usp3 Δ/Δ LSKs (D and E) or total BM cells (F). Representative LSKs images (D) and the Average Tail Moment calculated by Comet Score on LSKs (E) or BM cells (F) are shown. A minimum of 150 comets was evaluated per sample. n = 3 per genotype, 44 wk old. Bar, 50 µm. (G–J) Sorted LT-HSCs from BM of 40–44 wk old mice were grown in liquid cultures and analyzed for DNA damage. (G) Immunostaining of γH2AX and 53BP1 on LT-HSCs after 8–11 d in culture. The percentage of cells containing > 5 γH2AX and 53BP1 foci was evaluated in three independent experiments. n > 50 cells/genotype/experiment. Arrows: γH2AX-53BP1 colocalizing foci. Bar, 5 µm. (H) Immunostaining of FK2 LT-HSCs after 8–11 d in culture. Representative images and quantification by Image J of FK2 signal intensity from three independent experiments. Nuclei are outlined. n > 100 cells/genotype/experiment. Bar, 10 µm. (I) Co-immunostaining of FK2 and 53BP1 on LT-HSCs after 8–11 d in culture. The number of co-foci (arrows) per cell was quantified in 2 independent experiments, on a total of n = 145 (WT) or 80 ( Usp3 Δ/Δ) cells scored. Bar, 10 µm. (J) Percentage of micronuclei in LT-HSCs cultures after 8 or 15 d in culture. A minimum of 70 cells/sample was scored in three independent experiments. Bars, 10 µm. In all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Results are mean ± SEM (B, E, F, H, and I) or mean ± SD (G and J).
    Figure Legend Snippet: USP3-deficient HSCs accumulate spontaneous DNA damage. (A–C) γH2AX immunostaining on sorted Lin − Sca1 + c-Kit + CD150 + flk2/CD135 − CD34 − (LT-HSC) or CD34 + (ST-HSC) from 44-wk-old (A and B) or 17-wk-old (C) mice. Representative images (A) of LT-HSCs and ST-HSCs from 44-wk-old mice and quantification of the number of γH2AX foci/cell in HSCs from 44-wk-old (B) or 17-wk-old mice (minimum of 200 cells per genotype; C). Results are from two independent experiments. n = 3 mice/genotype/experiment. Bar, 5 µm. (D–F) Alkaline comet assay on sorted Usp3 Δ/Δ LSKs (D and E) or total BM cells (F). Representative LSKs images (D) and the Average Tail Moment calculated by Comet Score on LSKs (E) or BM cells (F) are shown. A minimum of 150 comets was evaluated per sample. n = 3 per genotype, 44 wk old. Bar, 50 µm. (G–J) Sorted LT-HSCs from BM of 40–44 wk old mice were grown in liquid cultures and analyzed for DNA damage. (G) Immunostaining of γH2AX and 53BP1 on LT-HSCs after 8–11 d in culture. The percentage of cells containing > 5 γH2AX and 53BP1 foci was evaluated in three independent experiments. n > 50 cells/genotype/experiment. Arrows: γH2AX-53BP1 colocalizing foci. Bar, 5 µm. (H) Immunostaining of FK2 LT-HSCs after 8–11 d in culture. Representative images and quantification by Image J of FK2 signal intensity from three independent experiments. Nuclei are outlined. n > 100 cells/genotype/experiment. Bar, 10 µm. (I) Co-immunostaining of FK2 and 53BP1 on LT-HSCs after 8–11 d in culture. The number of co-foci (arrows) per cell was quantified in 2 independent experiments, on a total of n = 145 (WT) or 80 ( Usp3 Δ/Δ) cells scored. Bar, 10 µm. (J) Percentage of micronuclei in LT-HSCs cultures after 8 or 15 d in culture. A minimum of 70 cells/sample was scored in three independent experiments. Bars, 10 µm. In all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Results are mean ± SEM (B, E, F, H, and I) or mean ± SD (G and J).

    Techniques Used: Immunostaining, Mouse Assay, Alkaline Single Cell Gel Electrophoresis

    Reduced size of adult HSC and CLP compartments and impaired pre–B lymphoid colony-forming activity in vitro in Usp3 Δ/Δ mice. (A–C) Multiparameter flow cytometry analysis of primitive hematopoietic populations. Gating strategies and representative FACS profiles are presented in Fig. S2 . (A) Absolute cell numbers of primitive populations from BM (2 femurs and 2 hips bones) of WT and Usp3 Δ/Δ mice: LSK (Lin − Sca1 + cKit + ), LT-HSC (LSK, flk2/CD135 − , CD150 + , CD34 − , LT-HSC), and ST-HSC (LSK, flk2/CD135 − , CD150 + , CD34 + , ST-HSC). Mean ± SEM is shown. (B and C) Frequency of LSKs, LT-HSCs, ST-HSCs, and MPPs (B) or CLPs, CMPs, GMPs, and MEPs (C) in BM of Usp3 Δ/Δ mice was calculated and normalized relative to WT animals. Mean ± SD is shown. (A–C) Results are from two (17 wk) or three (44 wk) independent experiments. 17 wk, n = 5 per genotype; 44 wk, n = 11 per genotype. (D and E) BM cells from WT or Usp3 Δ/Δ mice were assayed for pre–B (D) or myeloid colony-forming (CFU-C; E) ability. Results are from at least two independent experiments, n = 3 per group per experiment. Mean ± SD is shown. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, not significant.
    Figure Legend Snippet: Reduced size of adult HSC and CLP compartments and impaired pre–B lymphoid colony-forming activity in vitro in Usp3 Δ/Δ mice. (A–C) Multiparameter flow cytometry analysis of primitive hematopoietic populations. Gating strategies and representative FACS profiles are presented in Fig. S2 . (A) Absolute cell numbers of primitive populations from BM (2 femurs and 2 hips bones) of WT and Usp3 Δ/Δ mice: LSK (Lin − Sca1 + cKit + ), LT-HSC (LSK, flk2/CD135 − , CD150 + , CD34 − , LT-HSC), and ST-HSC (LSK, flk2/CD135 − , CD150 + , CD34 + , ST-HSC). Mean ± SEM is shown. (B and C) Frequency of LSKs, LT-HSCs, ST-HSCs, and MPPs (B) or CLPs, CMPs, GMPs, and MEPs (C) in BM of Usp3 Δ/Δ mice was calculated and normalized relative to WT animals. Mean ± SD is shown. (A–C) Results are from two (17 wk) or three (44 wk) independent experiments. 17 wk, n = 5 per genotype; 44 wk, n = 11 per genotype. (D and E) BM cells from WT or Usp3 Δ/Δ mice were assayed for pre–B (D) or myeloid colony-forming (CFU-C; E) ability. Results are from at least two independent experiments, n = 3 per group per experiment. Mean ± SD is shown. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ns, not significant.

    Techniques Used: Activity Assay, In Vitro, Mouse Assay, Flow Cytometry, Cytometry, FACS

    Usp3 Δ/Δ mice exhibit shorter lifespan, increased tumorigenesis, and spontaneous genotoxic stress in MEFs. (A–E) Cohorts of WT ( n = 26) and Usp3 Δ/Δ ( n = 34) mice were monitored for survival for 90 wk. (A) Kaplan Meier general survival analysis. (B) Histopathological analysis of spleens from WT and Usp3 Δ/Δ mice and representative H E-stained spleen sections from 5-mo-old animals. Bars: (left) 500 µm; (right) 20 µm. a Low myelopoiesis in one 10-mo-old Usp3 Δ/Δ animal; b low lymphoid compartment in a 15-mo-old Usp3 Δ/Δ mouse. (C) Kaplan Meier tumor-free survival analysis and distribution of tumor types in Usp3 Δ/Δ mice. (D and E). H E staining of histological sections of representative malignancies in Usp3 Δ/Δ mice. (D) Moderately differentiated papillary carcinoma of the lung (17 mo). (E) Adenomatosis in the stomach (14 mo). Bars: (top) 500 µm; (bottom) 50 µm. (F) Constant field gel electrophoresis (CFGE) analysis of WT and Usp3 Δ/Δ MEFs. Results are the mean ± SD of three independent experiments. (G) Quantification of chromosomal aberrations in metaphase preparations of WT and Usp3 Δ/Δ MEF. A minimum of 42 cells/genotype was assessed. Mean ± SEM of one of two representative experiments is shown. (H) Metaphase analysis of WT and Usp3 Δ/Δ MEFs immortalized with p53 knockdown (sh-p53). Arrowheads: chromatid break, chromosome fragment, ring chromosome. Inset, chromatid break. Results are the mean ± SD of three independent experiments with a minimum of 30 metaphase/genotype each counted. Bar, 10 µm. (I) SCEs analysis in WT and Usp3 Δ/Δ sh-p53 MEFs. SCEs in representative metaphases are indicated by arrows. Inset, chromosome with double SCE. SCEs in a minimum of 48 cells/genotype were quantified. Mean ± SEM of one of two representative experiments is shown. Bar, 10 µm. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. P-value was assessed by Log-rank test (A and C) or by Student’s t test (F–I).
    Figure Legend Snippet: Usp3 Δ/Δ mice exhibit shorter lifespan, increased tumorigenesis, and spontaneous genotoxic stress in MEFs. (A–E) Cohorts of WT ( n = 26) and Usp3 Δ/Δ ( n = 34) mice were monitored for survival for 90 wk. (A) Kaplan Meier general survival analysis. (B) Histopathological analysis of spleens from WT and Usp3 Δ/Δ mice and representative H E-stained spleen sections from 5-mo-old animals. Bars: (left) 500 µm; (right) 20 µm. a Low myelopoiesis in one 10-mo-old Usp3 Δ/Δ animal; b low lymphoid compartment in a 15-mo-old Usp3 Δ/Δ mouse. (C) Kaplan Meier tumor-free survival analysis and distribution of tumor types in Usp3 Δ/Δ mice. (D and E). H E staining of histological sections of representative malignancies in Usp3 Δ/Δ mice. (D) Moderately differentiated papillary carcinoma of the lung (17 mo). (E) Adenomatosis in the stomach (14 mo). Bars: (top) 500 µm; (bottom) 50 µm. (F) Constant field gel electrophoresis (CFGE) analysis of WT and Usp3 Δ/Δ MEFs. Results are the mean ± SD of three independent experiments. (G) Quantification of chromosomal aberrations in metaphase preparations of WT and Usp3 Δ/Δ MEF. A minimum of 42 cells/genotype was assessed. Mean ± SEM of one of two representative experiments is shown. (H) Metaphase analysis of WT and Usp3 Δ/Δ MEFs immortalized with p53 knockdown (sh-p53). Arrowheads: chromatid break, chromosome fragment, ring chromosome. Inset, chromatid break. Results are the mean ± SD of three independent experiments with a minimum of 30 metaphase/genotype each counted. Bar, 10 µm. (I) SCEs analysis in WT and Usp3 Δ/Δ sh-p53 MEFs. SCEs in representative metaphases are indicated by arrows. Inset, chromosome with double SCE. SCEs in a minimum of 48 cells/genotype were quantified. Mean ± SEM of one of two representative experiments is shown. Bar, 10 µm. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. P-value was assessed by Log-rank test (A and C) or by Student’s t test (F–I).

    Techniques Used: Mouse Assay, Staining, Nucleic Acid Electrophoresis

    USP3-deficient mice develop lymphopenia with age. (A) Peripheral blood cell counts in aged (44 wk old) WT and Usp3 Δ/Δ mice. B220 + , B lymphocytes; CD3 + , T lymphocytes; CD11b + , monocytes, granulocytes, and macrophages. Data are means ± SD. WT, n = 7; Usp3 Δ/Δ, n = 7. Representative FACS profiles are shown in Fig. S1 . (B) Flow cytometry analysis of BM of aged WT and Usp3 Δ/Δ mice for lymphoid (CD19 +/low ) and myeloid (CD11b + ) cell populations. Cell numbers per BM (2 femurs) are shown. Data are means ± SEM. WT, n = 10; Usp3 Δ/Δ, n = 10. (C) Flow cytometry analysis of B cell differentiation in the BM of aged WT and Usp3 Δ/Δ mice: Pre–B (B220 low IgM − cKit − CD25 + ), Pro–B (B220 low IgM − cKit + CD25 − ), immature B (B220 low IgM + ), and mature B (B220 high IgM + ) cells. Cell numbers per BM (2 femurs) are shown. Data are means ± SD. WT, n = 8; Usp3 Δ/Δ, n = 7. (D) Frequency (percentage of total B220 + B cell population) of the B cell subsets analyzed in C. Results are from two (A, C, and D) or three (B) independent experiments. For all panels: **, P ≤ 0.01; ns, not significant.
    Figure Legend Snippet: USP3-deficient mice develop lymphopenia with age. (A) Peripheral blood cell counts in aged (44 wk old) WT and Usp3 Δ/Δ mice. B220 + , B lymphocytes; CD3 + , T lymphocytes; CD11b + , monocytes, granulocytes, and macrophages. Data are means ± SD. WT, n = 7; Usp3 Δ/Δ, n = 7. Representative FACS profiles are shown in Fig. S1 . (B) Flow cytometry analysis of BM of aged WT and Usp3 Δ/Δ mice for lymphoid (CD19 +/low ) and myeloid (CD11b + ) cell populations. Cell numbers per BM (2 femurs) are shown. Data are means ± SEM. WT, n = 10; Usp3 Δ/Δ, n = 10. (C) Flow cytometry analysis of B cell differentiation in the BM of aged WT and Usp3 Δ/Δ mice: Pre–B (B220 low IgM − cKit − CD25 + ), Pro–B (B220 low IgM − cKit + CD25 − ), immature B (B220 low IgM + ), and mature B (B220 high IgM + ) cells. Cell numbers per BM (2 femurs) are shown. Data are means ± SD. WT, n = 8; Usp3 Δ/Δ, n = 7. (D) Frequency (percentage of total B220 + B cell population) of the B cell subsets analyzed in C. Results are from two (A, C, and D) or three (B) independent experiments. For all panels: **, P ≤ 0.01; ns, not significant.

    Techniques Used: Mouse Assay, FACS, Flow Cytometry, Cytometry, Cell Differentiation

    Usp3 Δ/Δ mice are viable. (A) Generation of conditional ( Usp3 Lox ) and null ( Usp3 Δ) USP3 alleles. USP3 protein domains and gene locus are schematically represented. ZnF, zinc finger Ub binding domain (ZnF-UBP); USP, Ub-specific protease domain. The targeting construct for Usp3 (thick blue line) contains LoxP (L, red triangles) sites positioned in introns flanking exon 2 and 3. Numbered gray boxes: exons. Triangles: FRT (F) sites. Puro: puromycin Dtk selection cassette. Restriction enzymes used for screening: B, BamHI; E, EcoRI; K, KpnI. Thick black lines: DNA probes used in Southern blot analysis. (B) PCR analysis of genomic DNA isolated from targeted ES clones. (C–G) Actin-Cre deleter strain was used for germline deletion and intercrossing of Usp3 Δ/+ mice produced Usp3 Δ/Δ homozygous animals, confirmed by PCR analysis (C) and Southern blot (D) on tail tip DNA. (E) Genotype frequency per litter, on a total of 24 litters. n = number of born mice/genotype. Mean ± SD is shown. (F) Immunoblot of whole cell extract (WCE) from tissues from WT and Usp3 Δ/Δ mice with anti-USP3 and anti-CDK4 antibody. (G) Reverse transcription qPCR analysis of the relative expression of USP3 transcript in WT and Usp3 Δ/Δ MEFs (mouse embryonic fibroblasts).
    Figure Legend Snippet: Usp3 Δ/Δ mice are viable. (A) Generation of conditional ( Usp3 Lox ) and null ( Usp3 Δ) USP3 alleles. USP3 protein domains and gene locus are schematically represented. ZnF, zinc finger Ub binding domain (ZnF-UBP); USP, Ub-specific protease domain. The targeting construct for Usp3 (thick blue line) contains LoxP (L, red triangles) sites positioned in introns flanking exon 2 and 3. Numbered gray boxes: exons. Triangles: FRT (F) sites. Puro: puromycin Dtk selection cassette. Restriction enzymes used for screening: B, BamHI; E, EcoRI; K, KpnI. Thick black lines: DNA probes used in Southern blot analysis. (B) PCR analysis of genomic DNA isolated from targeted ES clones. (C–G) Actin-Cre deleter strain was used for germline deletion and intercrossing of Usp3 Δ/+ mice produced Usp3 Δ/Δ homozygous animals, confirmed by PCR analysis (C) and Southern blot (D) on tail tip DNA. (E) Genotype frequency per litter, on a total of 24 litters. n = number of born mice/genotype. Mean ± SD is shown. (F) Immunoblot of whole cell extract (WCE) from tissues from WT and Usp3 Δ/Δ mice with anti-USP3 and anti-CDK4 antibody. (G) Reverse transcription qPCR analysis of the relative expression of USP3 transcript in WT and Usp3 Δ/Δ MEFs (mouse embryonic fibroblasts).

    Techniques Used: Mouse Assay, Binding Assay, Construct, Selection, Southern Blot, Polymerase Chain Reaction, Isolation, Clone Assay, Produced, Real-time Polymerase Chain Reaction, Expressing

    USP3-deficient HSCs have a cell-autonomous defect in repopulating ability in vivo and in colony formation in vitro. (A) Competitive transplantation of BM cells from 8-wk-old WT or Usp3 Δ/Δ (CD45.2; test) mice with WT (CD45.1; support) BM cells showing total reconstitution (left) and contribution of donor-derived cells to B cell (B220 + ), T cell (CD3 + ), and myeloid (Gr1 + ) lineages (middle) in the blood, or to LSKs in the BM (right) of irradiated recipients at the indicated wpt. Data are mean ± SD ( n = 5 per genotype). One of two representative experiments is shown. PBC, peripheral blood cell. (B) Noncompetitive transplantation of BM cells from aged (39–42 wk old) WT or Usp3 Δ/Δ mice. Donor-derived Lin − , LSKs, and HSCs in primary recipients at 16 wpt is shown. Data are mean ± SD ( n = 5 per genotype). (C) WT or Usp3 Δ/Δ BM cells from 8-wk-old mice were used in noncompetitive serial transplantations. Donor-derived LSKs in the BM of secondary recipients (separated by a 12 wk reconstitution period) are shown. Data are mean ± SEM ( n = 5 per genotype). (D) Total BM cell numbers in WT or Usp3 Δ/Δ mice at 17 and 44 wk of age (WT = 5, Usp3 Δ/Δ = 6, in two independent experiments; 2 femurs and 2 hip bones) or in 44-wk-old mice ( n = 3 per genotype) upon 5-FU treatment (2 femurs). Data are mean ± SEM. (E) LTC-IC assay using WT or Usp3 Δ/Δ Lin − cells purified from 8–16-wk-old mice (three experiments, n = 4 mice/genotype/experiment). The number of LSKs in the Lin − populations was evaluated by phenotypic profiling before plating, and results are expressed as total number of CFU-C normalized to 2,000 LSK plated. Data are mean ± SEM. In all BM transplantations, BM cells corresponding to stem cell equivalents were transplanted. In B and C, BM cells from n = 3 donor mice per genotype were pooled before primary transplantation. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
    Figure Legend Snippet: USP3-deficient HSCs have a cell-autonomous defect in repopulating ability in vivo and in colony formation in vitro. (A) Competitive transplantation of BM cells from 8-wk-old WT or Usp3 Δ/Δ (CD45.2; test) mice with WT (CD45.1; support) BM cells showing total reconstitution (left) and contribution of donor-derived cells to B cell (B220 + ), T cell (CD3 + ), and myeloid (Gr1 + ) lineages (middle) in the blood, or to LSKs in the BM (right) of irradiated recipients at the indicated wpt. Data are mean ± SD ( n = 5 per genotype). One of two representative experiments is shown. PBC, peripheral blood cell. (B) Noncompetitive transplantation of BM cells from aged (39–42 wk old) WT or Usp3 Δ/Δ mice. Donor-derived Lin − , LSKs, and HSCs in primary recipients at 16 wpt is shown. Data are mean ± SD ( n = 5 per genotype). (C) WT or Usp3 Δ/Δ BM cells from 8-wk-old mice were used in noncompetitive serial transplantations. Donor-derived LSKs in the BM of secondary recipients (separated by a 12 wk reconstitution period) are shown. Data are mean ± SEM ( n = 5 per genotype). (D) Total BM cell numbers in WT or Usp3 Δ/Δ mice at 17 and 44 wk of age (WT = 5, Usp3 Δ/Δ = 6, in two independent experiments; 2 femurs and 2 hip bones) or in 44-wk-old mice ( n = 3 per genotype) upon 5-FU treatment (2 femurs). Data are mean ± SEM. (E) LTC-IC assay using WT or Usp3 Δ/Δ Lin − cells purified from 8–16-wk-old mice (three experiments, n = 4 mice/genotype/experiment). The number of LSKs in the Lin − populations was evaluated by phenotypic profiling before plating, and results are expressed as total number of CFU-C normalized to 2,000 LSK plated. Data are mean ± SEM. In all BM transplantations, BM cells corresponding to stem cell equivalents were transplanted. In B and C, BM cells from n = 3 donor mice per genotype were pooled before primary transplantation. For all panels: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

    Techniques Used: In Vivo, In Vitro, Transplantation Assay, Mouse Assay, Derivative Assay, Irradiation, Purification

    24) Product Images from "Muller's Ratchet and compensatory mutation in Caenorhabditis briggsae mitochondrial genome evolution"

    Article Title: Muller's Ratchet and compensatory mutation in Caenorhabditis briggsae mitochondrial genome evolution

    Journal: BMC Evolutionary Biology

    doi: 10.1186/1471-2148-8-62

    ψND5 element positions in C. briggsae mitochondrial genomes . Dashed boxes indicate the two ψND5 elements and open boxes indicate mtDNA genes. Protein-coding genes are indicated by their common abbreviations and tRNA genes are indicated by their respective associated single-letter amino acid codes. ψND5-1 is 214–223 bp, depending on isolate, and ψND5-2 is 325–344 bp. The dashed horizontal line on top indicates DNA sequences that are lost in heteroplasmic ND5 deletion variants and the arrows indicate the primer positions that are employed for conventional PCR assays.
    Figure Legend Snippet: ψND5 element positions in C. briggsae mitochondrial genomes . Dashed boxes indicate the two ψND5 elements and open boxes indicate mtDNA genes. Protein-coding genes are indicated by their common abbreviations and tRNA genes are indicated by their respective associated single-letter amino acid codes. ψND5-1 is 214–223 bp, depending on isolate, and ψND5-2 is 325–344 bp. The dashed horizontal line on top indicates DNA sequences that are lost in heteroplasmic ND5 deletion variants and the arrows indicate the primer positions that are employed for conventional PCR assays.

    Techniques Used: Polymerase Chain Reaction

    25) Product Images from "Multiple Sodium Channel Variants in the Mosquito Culex quinquefasciatus"

    Article Title: Multiple Sodium Channel Variants in the Mosquito Culex quinquefasciatus

    Journal: International Journal of Biological Sciences

    doi: 10.7150/ijbs.4966

    Alternative splicing of Cx-Nav from mosquitoes Culex quinquefasciatus . Boxes represent exons. The junctions of exons are indicated with straight lines or bridge lines. The schematic of the predicted 6 segments (S1 to S6) in each of the 4 domains (I, II, III, and IV) in the structure of Cx-Nav protein are shown. *The transcript had an entire ORF.
    Figure Legend Snippet: Alternative splicing of Cx-Nav from mosquitoes Culex quinquefasciatus . Boxes represent exons. The junctions of exons are indicated with straight lines or bridge lines. The schematic of the predicted 6 segments (S1 to S6) in each of the 4 domains (I, II, III, and IV) in the structure of Cx-Nav protein are shown. *The transcript had an entire ORF.

    Techniques Used:

    26) Product Images from "Endosymbiotic Gene Transfer in Tertiary Plastid-Containing Dinoflagellates"

    Article Title: Endosymbiotic Gene Transfer in Tertiary Plastid-Containing Dinoflagellates

    Journal: Eukaryotic Cell

    doi: 10.1128/EC.00299-13

    (A) Total GC content and GC content at third codon positions (GC3) for the 17 genes with diatom affinity in dinotoms. The sequence names are abbreviated as follows: Db, D. baltica ; Kf, K. foliaceum . (B) Frequency distribution of the GC content for all transcripts from F. cylindrus , P. tricornutum , and T. pseudonana and all contigs from the D. baltica SL library. Gray arrows denote the genes with diatom affinity with low GC content; pink arrows denote the genes with diatom affinity with high GC content.
    Figure Legend Snippet: (A) Total GC content and GC content at third codon positions (GC3) for the 17 genes with diatom affinity in dinotoms. The sequence names are abbreviated as follows: Db, D. baltica ; Kf, K. foliaceum . (B) Frequency distribution of the GC content for all transcripts from F. cylindrus , P. tricornutum , and T. pseudonana and all contigs from the D. baltica SL library. Gray arrows denote the genes with diatom affinity with low GC content; pink arrows denote the genes with diatom affinity with high GC content.

    Techniques Used: Sequencing

    27) Product Images from "Genetic variants and cellular stressors associated with exfoliation syndrome modulate promoter activity of a lncRNA within the LOXL1 locus"

    Article Title: Genetic variants and cellular stressors associated with exfoliation syndrome modulate promoter activity of a lncRNA within the LOXL1 locus

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddv347

    Dual-luciferase reporter assay to test for promoter activity in the LOXL1 intron 1 region. ( A ) To test the hypothesis that the highly associated ∼7-kb region at the LOXL1 exon 1/intron 1 boundary contains a promoter for the LOXL1 antisense RNA
    Figure Legend Snippet: Dual-luciferase reporter assay to test for promoter activity in the LOXL1 intron 1 region. ( A ) To test the hypothesis that the highly associated ∼7-kb region at the LOXL1 exon 1/intron 1 boundary contains a promoter for the LOXL1 antisense RNA

    Techniques Used: Luciferase, Reporter Assay, Activity Assay

    DNase I hypersensitivity site mapping in the LOXL1 / LOXL1-AS1 genomic region in ocular cells. DNase I hypersensitivity mapping was used to query the region of interest at the LOXL1 exon 1/intron 1 boundary for evidence of regulatory elements. Cells from
    Figure Legend Snippet: DNase I hypersensitivity site mapping in the LOXL1 / LOXL1-AS1 genomic region in ocular cells. DNase I hypersensitivity mapping was used to query the region of interest at the LOXL1 exon 1/intron 1 boundary for evidence of regulatory elements. Cells from

    Techniques Used:

    Identification of a novel LOXL1-AS1 isoform. Forward and reverse primers (arrowheads) were designed to amplify a known LOXL1-AS1 isoform (ENST00000566011). This isoform was expressed in a dermal fibroblast cell line (NHDF-Ad, Lonza), resulting in a 2099-bp
    Figure Legend Snippet: Identification of a novel LOXL1-AS1 isoform. Forward and reverse primers (arrowheads) were designed to amplify a known LOXL1-AS1 isoform (ENST00000566011). This isoform was expressed in a dermal fibroblast cell line (NHDF-Ad, Lonza), resulting in a 2099-bp

    Techniques Used:

    Effects of Ocular Cell Stressors on LOXL1-AS1 gene expression. ( A ) To test for the effects of oxidative stress on LOXL1-AS1 expression, B-3 human LE cells were treated with 100, 250 or 500 μ m H 2 O 2 . A dose–response relationship was seen,
    Figure Legend Snippet: Effects of Ocular Cell Stressors on LOXL1-AS1 gene expression. ( A ) To test for the effects of oxidative stress on LOXL1-AS1 expression, B-3 human LE cells were treated with 100, 250 or 500 μ m H 2 O 2 . A dose–response relationship was seen,

    Techniques Used: Expressing

    Effects of XFS-associated alleles on LOXL1-AS1 promoter activity. The highest promoter activity was seen in the construct containing 1240 bp of sequence upstream of the LOXL1-AS1 start site (Fig. ). This −1240-bp construct contains
    Figure Legend Snippet: Effects of XFS-associated alleles on LOXL1-AS1 promoter activity. The highest promoter activity was seen in the construct containing 1240 bp of sequence upstream of the LOXL1-AS1 start site (Fig. ). This −1240-bp construct contains

    Techniques Used: Activity Assay, Construct, Sequencing

    LocusZoom plot of association in a South African XFS data set. The entire LOXL1 genomic region (∼40 kb) was sequenced in a data set of 50 black South African XFS cases and 50 age- and gender-matched controls. This region included all LOXL1 and
    Figure Legend Snippet: LocusZoom plot of association in a South African XFS data set. The entire LOXL1 genomic region (∼40 kb) was sequenced in a data set of 50 black South African XFS cases and 50 age- and gender-matched controls. This region included all LOXL1 and

    Techniques Used:

    28) Product Images from "Polyclonality of BRAF Mutations in Acquired Melanocytic Nevi"

    Article Title: Polyclonality of BRAF Mutations in Acquired Melanocytic Nevi

    Journal: JNCI Journal of the National Cancer Institute

    doi: 10.1093/jnci/djp309

    Polyclonality of v-raf murine sarcoma viral oncogene homolog B1 ( BRAF ) mutations in acquired melanocytic nevi. A ) Selection of single nevus cells after immunomagnetic separation. Single nevus cells ( purple dots with arrows ) were captured by high molecular weight-melanoma-associated antigen–specific monoclonal antibodies bound to immunomagnetic beads ( pink dots ). The cells ( encircled ) were procured by laser-capture microdissection ( top ; bar = 20 μm). Polymerase chain reaction (PCR) amplification and subsequent sequencing of single nevus cells showed wild-type BRAF and BRAF V600E mutations ( bottom ). B ) Laser-capture microdissection of frozen tissue section of acquired melanocytic nevi followed by direct sequencing of BRAF exon 15 ( top ; bar = 20 μm). Sequencing revealed two of the contiguous single nevus cells to have the BRAF V600E mutation and one to have a compound heterozygous BRAF V600E (T1799A) and BRAF V600G (T1799G) mutation, showing a heterogeneous pattern of BRAF mutations in proximal cells on a single-cell level ( bottom ). C ) Subcloning and subsequent sequencing of BRAF exon 15 and the single nucleotide polymorphism (SNP) rs7801086. This SNP maps approximately 2 kb telomeric from BRAF exon 15. Four nevi (numbers 3, 6, 11, and 14) were excised from patients who were heterozygous for this SNP. DNA was extracted from hundreds of nevus cells isolated either by using immunomagnetic beads (numbers 3 and 6) or laser-capture microdissection of frozen tissue sections (numbers 11 and 14). A 2859-bp fragment containing both BRAF exon 15 and the SNP rs7801086 was amplified by long-range PCR. Subcloning was carried out using this fragment as an insert. Sixteen to 30 colonies were randomly picked from each patient sample and analyzed for the sequence of both BRAF exon 15 and rs7801086. In all four patient samples, colonies with BRAF V600E as well as wild-type BRAF were accompanied by different SNP alleles, some harboring the G allele and others harboring the T allele. In sample number 14, one colony (*) showed a tandem BRAF V600E/K601E (T1799→A and A1802→G) mutation.
    Figure Legend Snippet: Polyclonality of v-raf murine sarcoma viral oncogene homolog B1 ( BRAF ) mutations in acquired melanocytic nevi. A ) Selection of single nevus cells after immunomagnetic separation. Single nevus cells ( purple dots with arrows ) were captured by high molecular weight-melanoma-associated antigen–specific monoclonal antibodies bound to immunomagnetic beads ( pink dots ). The cells ( encircled ) were procured by laser-capture microdissection ( top ; bar = 20 μm). Polymerase chain reaction (PCR) amplification and subsequent sequencing of single nevus cells showed wild-type BRAF and BRAF V600E mutations ( bottom ). B ) Laser-capture microdissection of frozen tissue section of acquired melanocytic nevi followed by direct sequencing of BRAF exon 15 ( top ; bar = 20 μm). Sequencing revealed two of the contiguous single nevus cells to have the BRAF V600E mutation and one to have a compound heterozygous BRAF V600E (T1799A) and BRAF V600G (T1799G) mutation, showing a heterogeneous pattern of BRAF mutations in proximal cells on a single-cell level ( bottom ). C ) Subcloning and subsequent sequencing of BRAF exon 15 and the single nucleotide polymorphism (SNP) rs7801086. This SNP maps approximately 2 kb telomeric from BRAF exon 15. Four nevi (numbers 3, 6, 11, and 14) were excised from patients who were heterozygous for this SNP. DNA was extracted from hundreds of nevus cells isolated either by using immunomagnetic beads (numbers 3 and 6) or laser-capture microdissection of frozen tissue sections (numbers 11 and 14). A 2859-bp fragment containing both BRAF exon 15 and the SNP rs7801086 was amplified by long-range PCR. Subcloning was carried out using this fragment as an insert. Sixteen to 30 colonies were randomly picked from each patient sample and analyzed for the sequence of both BRAF exon 15 and rs7801086. In all four patient samples, colonies with BRAF V600E as well as wild-type BRAF were accompanied by different SNP alleles, some harboring the G allele and others harboring the T allele. In sample number 14, one colony (*) showed a tandem BRAF V600E/K601E (T1799→A and A1802→G) mutation.

    Techniques Used: Selection, Immunomagnetic Separation, Molecular Weight, Laser Capture Microdissection, Polymerase Chain Reaction, Amplification, Sequencing, Mutagenesis, Subcloning, Isolation

    29) Product Images from "Loss of Mammal-specific Tectorial Membrane Component Carcinoembryonic Antigen Cell Adhesion Molecule 16 (CEACAM16) Leads to Hearing Impairment at Low and High Frequencies *"

    Article Title: Loss of Mammal-specific Tectorial Membrane Component Carcinoembryonic Antigen Cell Adhesion Molecule 16 (CEACAM16) Leads to Hearing Impairment at Low and High Frequencies *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.320481

    Targeted disruption of the murine Ceacam16 gene. A , shown is targeting strategy. Structure of the wild-type allele ( top ), targeting construct ( middle ), and recombinant allele ( bottom ) is shown. The 6 exons of Ceacam16 are shown as boxes with the encoded domains indicated above (exons encoding IgV-like domains are red ; IgC-like domains are blue ). Except for BamHI, only restriction endonuclease sites relevant for cloning or probe generation are shown. Neo and tk expression cassettes, which can be used for the selection of homologous recombinants (G418- and ganciclovir-resistant) are shown as open boxes . Black arrows indicate the transcriptional direction. The vector sequence within the targeting plasmid is shown as a thin line . The positions and predicted sizes of the DNA fragments obtained after digestion with BamHI and hybridization with the 5′- ( blue ) and 3′-probes ( red ), respectively, are indicated. Positions of primers used for identification of ES cell clones with homologous recombination, genotyping, and detection of Ceacam16 transcripts are shown by half arrows . B , identification of targeted ES cell clones by long range PCR is shown. A total of three recombinant ES cell clones could be identified of 200 clones tested. C , Southern blot analysis is shown. Analysis of DNA from ES cell clones B6, C3, and D1 and from F2 offspring (#97, #98, #152) of a C3-derived chimeric founder after heterozygous mating of F1 mice demonstrated correct recombination events at 3′-region and the 5′-region of the targeted allele of Ceacam16 , respectively. The absence and presence of CEACAM16 mRNA and protein was demonstrated in adult ( > 20 weeks old) Ceacam16 −/− and Ceacam16 +/+ mice by RT-PCR ( D ) and Western blotting ( E ), respectively. As a control, β-actin-specific primers and antibodies were used. The mobility and size of marker DNA fragments and proteins are shown in the left margins . Wild-type (+/+), heterozygous (+/−), and homozygous genotypes (−/−) for the Ceacam16 knock-out allele are indicated above the lanes .
    Figure Legend Snippet: Targeted disruption of the murine Ceacam16 gene. A , shown is targeting strategy. Structure of the wild-type allele ( top ), targeting construct ( middle ), and recombinant allele ( bottom ) is shown. The 6 exons of Ceacam16 are shown as boxes with the encoded domains indicated above (exons encoding IgV-like domains are red ; IgC-like domains are blue ). Except for BamHI, only restriction endonuclease sites relevant for cloning or probe generation are shown. Neo and tk expression cassettes, which can be used for the selection of homologous recombinants (G418- and ganciclovir-resistant) are shown as open boxes . Black arrows indicate the transcriptional direction. The vector sequence within the targeting plasmid is shown as a thin line . The positions and predicted sizes of the DNA fragments obtained after digestion with BamHI and hybridization with the 5′- ( blue ) and 3′-probes ( red ), respectively, are indicated. Positions of primers used for identification of ES cell clones with homologous recombination, genotyping, and detection of Ceacam16 transcripts are shown by half arrows . B , identification of targeted ES cell clones by long range PCR is shown. A total of three recombinant ES cell clones could be identified of 200 clones tested. C , Southern blot analysis is shown. Analysis of DNA from ES cell clones B6, C3, and D1 and from F2 offspring (#97, #98, #152) of a C3-derived chimeric founder after heterozygous mating of F1 mice demonstrated correct recombination events at 3′-region and the 5′-region of the targeted allele of Ceacam16 , respectively. The absence and presence of CEACAM16 mRNA and protein was demonstrated in adult ( > 20 weeks old) Ceacam16 −/− and Ceacam16 +/+ mice by RT-PCR ( D ) and Western blotting ( E ), respectively. As a control, β-actin-specific primers and antibodies were used. The mobility and size of marker DNA fragments and proteins are shown in the left margins . Wild-type (+/+), heterozygous (+/−), and homozygous genotypes (−/−) for the Ceacam16 knock-out allele are indicated above the lanes .

    Techniques Used: Construct, Recombinant, Clone Assay, Expressing, Selection, Plasmid Preparation, Sequencing, Hybridization, Homologous Recombination, Polymerase Chain Reaction, Southern Blot, Derivative Assay, Mouse Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Marker, Knock-Out

    30) Product Images from "Expansion of a novel endogenous retrovirus throughout the pericentromeres of modern humans"

    Article Title: Expansion of a novel endogenous retrovirus throughout the pericentromeres of modern humans

    Journal: Genome Biology

    doi: 10.1186/s13059-015-0641-1

    Mapping of K222 proviruses in the human genome. (A) Schematic representation of the primer sets used to isolate K222 by PCR. The genomic structure of a centromeric provirus K111 is shown; the viral genes gag , pro , pol , env , and np9 , surrounded by LTRs, integrated into centromeric repeats (CER:D22Z3). The target site duplication of K111 GAATTC is indicated. The primers P1 and P2 bind CER:D22Z3. These primers were used in combination with primers that span the provirus genome. Arrows indicate the position and orientation of the primers; the number above indicates the nucleotide position they bind in reference to K111. Mapping to the 5′ end of the provirus was performed using the primer P1 and a set of HERV-K (HML-2) reverse primers. Mapping to the 3′ end of the provirus was performed with the reverse primer P2 and a set of HERV-K (HML-2) forward primers. (B, C) Isolation of K222 provirus. The sequence of K222 was detected by PCR from DNA of the cell lines H9 and HUT78, which lack K111 5′ end. Normal human DNA, containing K111, was used as a control for the PCR reaction. The number shown for each lane represents the primers. The gels show the amplification products of the 5′ mapping (B) or 3′ mapping (C) of centromeric proviruses in H9, HUT78, and normal human DNA using different combinations of primers. A molecular size ladder is indicated at the left. No amplification products were detected in H9 and HUT78 cell lines, in contrast to normal human DNA, when using the primer sets P1-982R, P1-2499R (B) , or primer sets P2-1965F, and P2-2641F (C) . An asterisk indicates a band that was shown by sequencing to be the result of non-specific amplification. Sequencing of the mapping products obtained from DNA of H9 and HUT78 cells reveals the sequence of K222.
    Figure Legend Snippet: Mapping of K222 proviruses in the human genome. (A) Schematic representation of the primer sets used to isolate K222 by PCR. The genomic structure of a centromeric provirus K111 is shown; the viral genes gag , pro , pol , env , and np9 , surrounded by LTRs, integrated into centromeric repeats (CER:D22Z3). The target site duplication of K111 GAATTC is indicated. The primers P1 and P2 bind CER:D22Z3. These primers were used in combination with primers that span the provirus genome. Arrows indicate the position and orientation of the primers; the number above indicates the nucleotide position they bind in reference to K111. Mapping to the 5′ end of the provirus was performed using the primer P1 and a set of HERV-K (HML-2) reverse primers. Mapping to the 3′ end of the provirus was performed with the reverse primer P2 and a set of HERV-K (HML-2) forward primers. (B, C) Isolation of K222 provirus. The sequence of K222 was detected by PCR from DNA of the cell lines H9 and HUT78, which lack K111 5′ end. Normal human DNA, containing K111, was used as a control for the PCR reaction. The number shown for each lane represents the primers. The gels show the amplification products of the 5′ mapping (B) or 3′ mapping (C) of centromeric proviruses in H9, HUT78, and normal human DNA using different combinations of primers. A molecular size ladder is indicated at the left. No amplification products were detected in H9 and HUT78 cell lines, in contrast to normal human DNA, when using the primer sets P1-982R, P1-2499R (B) , or primer sets P2-1965F, and P2-2641F (C) . An asterisk indicates a band that was shown by sequencing to be the result of non-specific amplification. Sequencing of the mapping products obtained from DNA of H9 and HUT78 cells reveals the sequence of K222.

    Techniques Used: Polymerase Chain Reaction, Isolation, Sequencing, Amplification

    ChIP analysis shows that K222 proviruses are found in pericentromeric regions. Quantitative PCR of K222 DNA, the centromeric 11-mer alphoid repeat of chromosome 21 (alphoidChr.21) DNA, and 5S ribosomal DNA immunoprecipitated by antibodies to CENPA, CENPB, H3K9Me3, or control IgG. (A) Compared to the control IgG fraction, K222 is enriched 50-fold in the H3K9Me3 fraction, but not in the centromeric CENPA and CENPB protein fractions. (B) The positive control, the alphoid Chr.21 , is enriched approximately 8-fold in each of the CENPA and CENPB fractions, and approximately 650-fold in the H3K9Me3 fraction. (C) The negative control, 5S ribosomal DNA pre sent in the q arm of chromosome 1, shows no significant enrichment with antibodies to CENPA, CENPB, or H3K9Me3. Graphs show the relative enrichment normalized to control IgG-precipitated fractions from three independent experiments. Asterisks indicate statistical significance: *** = P
    Figure Legend Snippet: ChIP analysis shows that K222 proviruses are found in pericentromeric regions. Quantitative PCR of K222 DNA, the centromeric 11-mer alphoid repeat of chromosome 21 (alphoidChr.21) DNA, and 5S ribosomal DNA immunoprecipitated by antibodies to CENPA, CENPB, H3K9Me3, or control IgG. (A) Compared to the control IgG fraction, K222 is enriched 50-fold in the H3K9Me3 fraction, but not in the centromeric CENPA and CENPB protein fractions. (B) The positive control, the alphoid Chr.21 , is enriched approximately 8-fold in each of the CENPA and CENPB fractions, and approximately 650-fold in the H3K9Me3 fraction. (C) The negative control, 5S ribosomal DNA pre sent in the q arm of chromosome 1, shows no significant enrichment with antibodies to CENPA, CENPB, or H3K9Me3. Graphs show the relative enrichment normalized to control IgG-precipitated fractions from three independent experiments. Asterisks indicate statistical significance: *** = P

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Immunoprecipitation, Positive Control, Negative Control

    Detection of K222 in human chromosomes. (A) K222 was detected by PCR using the set of primers K222F and K222bR in DNA from human/rodent hybrid cell lines, which carry only one specific human chromosome. K222 was found in chromosomes 1, 7, 12, 13, 14, 15, 18, 21, and 22. Other bands (for example the PCR products detected in chromosomes 17, 19, 20, X, and Y) were shown by sequencing to be the result of non-specific PCR amplification. (B) Quantitation of K222 copies by qPCR in human chromosomes. The number of K222 copies was calculated from 250 ng of DNA from human/rodent cells lines. Assuming that human cells have between 8 and 61 K222 copies, then we could estimate that about one copy of K222 is present in chromosomes 1, 18, 21, 22, and perhaps more than one in chromosome 12. Several copies of K222, however, exist in chromosomes 7, 13, 14, and 15.
    Figure Legend Snippet: Detection of K222 in human chromosomes. (A) K222 was detected by PCR using the set of primers K222F and K222bR in DNA from human/rodent hybrid cell lines, which carry only one specific human chromosome. K222 was found in chromosomes 1, 7, 12, 13, 14, 15, 18, 21, and 22. Other bands (for example the PCR products detected in chromosomes 17, 19, 20, X, and Y) were shown by sequencing to be the result of non-specific PCR amplification. (B) Quantitation of K222 copies by qPCR in human chromosomes. The number of K222 copies was calculated from 250 ng of DNA from human/rodent cells lines. Assuming that human cells have between 8 and 61 K222 copies, then we could estimate that about one copy of K222 is present in chromosomes 1, 18, 21, 22, and perhaps more than one in chromosome 12. Several copies of K222, however, exist in chromosomes 7, 13, 14, and 15.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Amplification, Quantitation Assay, Real-time Polymerase Chain Reaction

    K222 provirus in the genomes of Old World monkeys, primates and humans. (A) Phylogenetic neighbor-joining tree of K222 integration sequences amplified from the DNA of baboon, orangutan, gorilla, chimpanzee, and human. The tree is unrooted, with taxa arranged for a balanced shape. The tree was constructed using the Kimura 2-parameter model. The stability of branches was evaluated by bootstrap tests with 10,000 replications. The scale bars represent the nucleotide substitutions per sequence. (B) Nucleotide sequence alignment of K222 insertion sequences amplified from the genomes of Old World monkeys, primates, and humans. The sequences are compared to the olive baboon sequence, which is the oldest germline sequence. Dots indicate nucleotide similarities to the master sequence. Nucleotide substitutions are indicated in letters. Several nucleotide insertions can be seen in the sequence of K222 in the orangutan, but not other primates or humans (B) , which cause the divergence of the orangutan K222 in the phylogenetic tree (A) , suggesting that these insertions arose only during the evolution of modern orangutans.
    Figure Legend Snippet: K222 provirus in the genomes of Old World monkeys, primates and humans. (A) Phylogenetic neighbor-joining tree of K222 integration sequences amplified from the DNA of baboon, orangutan, gorilla, chimpanzee, and human. The tree is unrooted, with taxa arranged for a balanced shape. The tree was constructed using the Kimura 2-parameter model. The stability of branches was evaluated by bootstrap tests with 10,000 replications. The scale bars represent the nucleotide substitutions per sequence. (B) Nucleotide sequence alignment of K222 insertion sequences amplified from the genomes of Old World monkeys, primates, and humans. The sequences are compared to the olive baboon sequence, which is the oldest germline sequence. Dots indicate nucleotide similarities to the master sequence. Nucleotide substitutions are indicated in letters. Several nucleotide insertions can be seen in the sequence of K222 in the orangutan, but not other primates or humans (B) , which cause the divergence of the orangutan K222 in the phylogenetic tree (A) , suggesting that these insertions arose only during the evolution of modern orangutans.

    Techniques Used: Amplification, Construct, Sequencing

    Detection of K222 and recombinant K222/K111 sequences in individuals lacking the K111 5′ end. (A) Amplification of K222/K111 recombinant sequences. K222/K111 sequences were amplified with the primer 7972F and the primer P2, which binds to the K111 3′ flanking sequence (see Figure 2 ) in the DNA from individuals who lack the K111 5′ end (68, 90, and 95) and the cell line HUT78, which also lacks the K111 integration. As a positive control we used the DNA of individual 96, who is positive for K111 5′ end. (B) Amplification of K222 3′ integration. K222 was amplified with the primer 7972F and K222LTR-pCER:D22Z8R, the latter primer binding to the LTR-pCER:D22Z8 junction sequence present in K222, but not in K111. K111 3′ integration instead has a 5 bp sequence from the LTR and the target site duplication GAATTC not present in K222. Amplification of K222 3′ integration was seen in individuals having (96) or lacking (68, 90, and HUT78) the K111 5′ end. (C) Evolution of K222 and K222/K111 recombinant sequences in humans. A Bayesian inference tree of K222 and K222/K111 LTR sequences obtained by PCR in individuals lacking the K111 5′ end. The K222 sequences amplified are indicated with a K222 label. The tree reveals two different K222 LTR clades; K222 sequences similar to the K222 provirus (blue) and sequences that cluster to the K111 provirus (red). K222 sequences in individuals lacking the K111 5′ end clustering to K111 indicate the likely existence of K111 in the ancestral human lineage of those individuals. The K222/K111 recombinant clade (red) also suggests that K222 and K111 likely recombined by recombination/gene conversion during human evolution before K111 was lost from the lineage. Posterior probability values > 85 are shown for the best tree.
    Figure Legend Snippet: Detection of K222 and recombinant K222/K111 sequences in individuals lacking the K111 5′ end. (A) Amplification of K222/K111 recombinant sequences. K222/K111 sequences were amplified with the primer 7972F and the primer P2, which binds to the K111 3′ flanking sequence (see Figure 2 ) in the DNA from individuals who lack the K111 5′ end (68, 90, and 95) and the cell line HUT78, which also lacks the K111 integration. As a positive control we used the DNA of individual 96, who is positive for K111 5′ end. (B) Amplification of K222 3′ integration. K222 was amplified with the primer 7972F and K222LTR-pCER:D22Z8R, the latter primer binding to the LTR-pCER:D22Z8 junction sequence present in K222, but not in K111. K111 3′ integration instead has a 5 bp sequence from the LTR and the target site duplication GAATTC not present in K222. Amplification of K222 3′ integration was seen in individuals having (96) or lacking (68, 90, and HUT78) the K111 5′ end. (C) Evolution of K222 and K222/K111 recombinant sequences in humans. A Bayesian inference tree of K222 and K222/K111 LTR sequences obtained by PCR in individuals lacking the K111 5′ end. The K222 sequences amplified are indicated with a K222 label. The tree reveals two different K222 LTR clades; K222 sequences similar to the K222 provirus (blue) and sequences that cluster to the K111 provirus (red). K222 sequences in individuals lacking the K111 5′ end clustering to K111 indicate the likely existence of K111 in the ancestral human lineage of those individuals. The K222/K111 recombinant clade (red) also suggests that K222 and K111 likely recombined by recombination/gene conversion during human evolution before K111 was lost from the lineage. Posterior probability values > 85 are shown for the best tree.

    Techniques Used: Recombinant, Amplification, Sequencing, Positive Control, Binding Assay, Polymerase Chain Reaction

    K222 integrated into the primate germline after the divergence of New and Old World monkeys and expanded in copy number during the evolution of humans. (A) Genomic organization of centromeric K111 and K222 proviruses. The positions of the primers used to amplify K222 insertions by PCR and qPCR are indicated by arrows. (B) Detection of K222 from DNA of New and Old-World primates. K222 was detected by PCR with the primers K222F and K222bR in the baboon, orangutan, gorilla, chimpanzee, and human, but not in macaques, African green monkeys, and New World monkeys. Other bands (for example, the PCR products detected in mouse, hamster, and rhesus macaque) were shown by sequencing to be the result of non-specific PCR amplification. A phylogeny of New World monkeys, Old World monkeys, and hominoids (humans and apes) is shown. Estimated times of divergence are shown. MYA: million years ago. (C) Quantitation of K222 copies by qPCR in the genomes of Old World monkeys, humans, and a number of other primates. K222 is likely present as a single copy in the genomes of baboon, orangutan, gorilla and chimpanzee, while present in multiple copies in the human genome. The label of each species in (B) matches to the bars.
    Figure Legend Snippet: K222 integrated into the primate germline after the divergence of New and Old World monkeys and expanded in copy number during the evolution of humans. (A) Genomic organization of centromeric K111 and K222 proviruses. The positions of the primers used to amplify K222 insertions by PCR and qPCR are indicated by arrows. (B) Detection of K222 from DNA of New and Old-World primates. K222 was detected by PCR with the primers K222F and K222bR in the baboon, orangutan, gorilla, chimpanzee, and human, but not in macaques, African green monkeys, and New World monkeys. Other bands (for example, the PCR products detected in mouse, hamster, and rhesus macaque) were shown by sequencing to be the result of non-specific PCR amplification. A phylogeny of New World monkeys, Old World monkeys, and hominoids (humans and apes) is shown. Estimated times of divergence are shown. MYA: million years ago. (C) Quantitation of K222 copies by qPCR in the genomes of Old World monkeys, humans, and a number of other primates. K222 is likely present as a single copy in the genomes of baboon, orangutan, gorilla and chimpanzee, while present in multiple copies in the human genome. The label of each species in (B) matches to the bars.

    Techniques Used: Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Sequencing, Amplification, Quantitation Assay

    Detection of the K222 provirus in the genome of human cell lines by slot blot analysis. The DNA of human cell lines that were found to have or lack the 5′ end of K111 by PCR, and presumably contain the truncated K222 provirus, were screened for K111 and K222 by slot blot analyses. (A) Generation of K111 and K222-specific biotinylated probes. Probes were generated by PCR incorporation of biotin-labeled dCTP. The K111 probe is 422 bp long and spans the CER:D22Z3 flanking sequence and the beginning of the LTR of K111. The K222 probe is 464 bp long and covers the pCER:D22Z8 flanking sequence and pro gene of K222. (B) DNA from the B-cell lines BJAB (having the 5′ end of K111) and IRA (lacking the 5′ end) as observed by PCR, were screened for K111 and K222 virus by slot blotting. DNA was cross-linked to PVDF membranes and screened for K111 and K222 using biotinylated probes. The probes were detected by chemiluminescence with HRP-conjugated streptavidin. The K111 probe, which targets the 5′ end of genomic K111, reacted with the DNA of BJAB cells but not IRA cells, confirming the lack of the 5′ end of the viral genome in IRA cells. The K222 probe reacted with the DNA of both BJAB and IRA cells, confirming that both cell lines have provirus K222, which is truncated at the 5′ end. Mouse DNA served as a negative control, and plasmids containing either K111 or K222 genomes were used as positive controls. The K111 probe did not react with the K222 plasmid and vice versa.
    Figure Legend Snippet: Detection of the K222 provirus in the genome of human cell lines by slot blot analysis. The DNA of human cell lines that were found to have or lack the 5′ end of K111 by PCR, and presumably contain the truncated K222 provirus, were screened for K111 and K222 by slot blot analyses. (A) Generation of K111 and K222-specific biotinylated probes. Probes were generated by PCR incorporation of biotin-labeled dCTP. The K111 probe is 422 bp long and spans the CER:D22Z3 flanking sequence and the beginning of the LTR of K111. The K222 probe is 464 bp long and covers the pCER:D22Z8 flanking sequence and pro gene of K222. (B) DNA from the B-cell lines BJAB (having the 5′ end of K111) and IRA (lacking the 5′ end) as observed by PCR, were screened for K111 and K222 virus by slot blotting. DNA was cross-linked to PVDF membranes and screened for K111 and K222 using biotinylated probes. The probes were detected by chemiluminescence with HRP-conjugated streptavidin. The K111 probe, which targets the 5′ end of genomic K111, reacted with the DNA of BJAB cells but not IRA cells, confirming the lack of the 5′ end of the viral genome in IRA cells. The K222 probe reacted with the DNA of both BJAB and IRA cells, confirming that both cell lines have provirus K222, which is truncated at the 5′ end. Mouse DNA served as a negative control, and plasmids containing either K111 or K222 genomes were used as positive controls. The K111 probe did not react with the K222 plasmid and vice versa.

    Techniques Used: Dot Blot, Polymerase Chain Reaction, Generated, Labeling, Sequencing, Negative Control, Plasmid Preparation

    Absence of K111 5′ end in the genome of some cell lines. (A) Genomic structure of the K111 provirus. Arrows indicate the position of the primers P1 and P4, which amplify the 5′ integration of K111, and the primer/probe combination K111F, K111R, and K111P that specifically discriminates the K111 and K222 env gene from other HERV-K (HML-2) env sequences due to a 6 bp mutation [ 10 ]. (B) Detection of K111 5′ end insertions in human cell lines. The 5′ flanking K111 insertions were detected in all human cell lines tested in this study by PCR using the primers P1 and P4 [ 10 ], except for the DNA of cell lines H9, HUT78, H9/HTLVIII, and the IRA B-cell line. Arrows indicate individual K111 insertional polymorphisms. Integrity of the DNA was assessed by amplification of GAPDH (see lower gel). The molecular size of the DNA ladder is shown on the left of the gel. On top of each lane is the name of each cell line subjected to study. The weak bands observed in H9 and H9/HTLVIII were shown by sequencing to be the result of non-specific PCR amplification.
    Figure Legend Snippet: Absence of K111 5′ end in the genome of some cell lines. (A) Genomic structure of the K111 provirus. Arrows indicate the position of the primers P1 and P4, which amplify the 5′ integration of K111, and the primer/probe combination K111F, K111R, and K111P that specifically discriminates the K111 and K222 env gene from other HERV-K (HML-2) env sequences due to a 6 bp mutation [ 10 ]. (B) Detection of K111 5′ end insertions in human cell lines. The 5′ flanking K111 insertions were detected in all human cell lines tested in this study by PCR using the primers P1 and P4 [ 10 ], except for the DNA of cell lines H9, HUT78, H9/HTLVIII, and the IRA B-cell line. Arrows indicate individual K111 insertional polymorphisms. Integrity of the DNA was assessed by amplification of GAPDH (see lower gel). The molecular size of the DNA ladder is shown on the left of the gel. On top of each lane is the name of each cell line subjected to study. The weak bands observed in H9 and H9/HTLVIII were shown by sequencing to be the result of non-specific PCR amplification.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Amplification, Sequencing

    Detection of K111 and K222 in the human population. (A) Genomic organization of K111 and K222 proviruses. The location of the primers to map K111 and K222 is shown. (B) Detection of K111 5′ end in the human population. The 5′ end of K111 was detected using the primers P1 and P4. The black arrow A indicates the K111 5′ end. The gray arrow indicates non-specific PCR products. On top of each lane is a number signifying each individual subjected to study. (C, D) Mapping of K111 (C) and K222 (D) in five individuals, who are positive or negative for the K111 5′ end, respectively. K111 mapping (C) was carried out with primer P1 and reverse primers that bind at positions 982, 2499, and 3460 bp of a K111 provirus. Black arrows indicate specific K111 insertions; A (product P1-982R), C (product P1-2499R), and D (product P1-3460R). The gray arrow indicates non-specific PCR amplifications. K111 detection was observed in the individuals labeled with the numbers, 1, 2, 3, 5, and 6, which are positive for the 5′ K111 end. Non-specific PCR product was detected in individuals labeled with the numbers 4, 68, 86, 90, and 95, which are negative for the 5′ K111 end as shown in B. The primers P1 and 3460R also detect K222 in individuals either negative or positive for the 5′ K111 integration (see stars). K222 mapping was carried out with the primer K222F and reverse primers that bind at positions 982, 1968, 2499, and 3460 bp in reference to K111. PCR products A, B, and C (black arrows) seen in the DNA of K111 positive individuals were shown to be the amplification of K111. No amplification products were seen in individuals lacking the 5′ end of K111. D represents the amplification product of K222.
    Figure Legend Snippet: Detection of K111 and K222 in the human population. (A) Genomic organization of K111 and K222 proviruses. The location of the primers to map K111 and K222 is shown. (B) Detection of K111 5′ end in the human population. The 5′ end of K111 was detected using the primers P1 and P4. The black arrow A indicates the K111 5′ end. The gray arrow indicates non-specific PCR products. On top of each lane is a number signifying each individual subjected to study. (C, D) Mapping of K111 (C) and K222 (D) in five individuals, who are positive or negative for the K111 5′ end, respectively. K111 mapping (C) was carried out with primer P1 and reverse primers that bind at positions 982, 2499, and 3460 bp of a K111 provirus. Black arrows indicate specific K111 insertions; A (product P1-982R), C (product P1-2499R), and D (product P1-3460R). The gray arrow indicates non-specific PCR amplifications. K111 detection was observed in the individuals labeled with the numbers, 1, 2, 3, 5, and 6, which are positive for the 5′ K111 end. Non-specific PCR product was detected in individuals labeled with the numbers 4, 68, 86, 90, and 95, which are negative for the 5′ K111 end as shown in B. The primers P1 and 3460R also detect K222 in individuals either negative or positive for the 5′ K111 integration (see stars). K222 mapping was carried out with the primer K222F and reverse primers that bind at positions 982, 1968, 2499, and 3460 bp in reference to K111. PCR products A, B, and C (black arrows) seen in the DNA of K111 positive individuals were shown to be the amplification of K111. No amplification products were seen in individuals lacking the 5′ end of K111. D represents the amplification product of K222.

    Techniques Used: Polymerase Chain Reaction, Labeling, Amplification

    31) Product Images from "The physiological level of rNMPs present in mtDNA does not compromise its stability"

    Article Title: The physiological level of rNMPs present in mtDNA does not compromise its stability

    Journal: bioRxiv

    doi: 10.1101/746719

    Deletion of SAMHD1 does not affect mtDNA stability. a) MtDNA copy number in the TA muscle of 5 or 6 wt (filled dots) and SAMHD1 −/− (open dots) 13-week-old (adult), 1-year-old (old adult), and 2-year-old (aged) animals was determined by qPCR and normalized to the value for adult wt mice. The mean for each group is indicated by a horizontal line. The p-values were calculated using Welch’s t-test; ns, non-significant. b) DNA isolated from embryos and from the TA muscle of pups, adults, 1-year-old (old) adults, or aged animals was linearized with SacI endonuclease and separated on a neutral gel. MtDNA was visualized as above. Full-length mtDNA is indicated (FL); the asterisk denotes a higher-migrating species resistant to cleavage. c) Long-range PCR to detect deletions in mtDNA from the TA muscle of wt mice of various ages. Full-length product is indicated (FL). Only minor species containing deletions are observed in the mtDNA from old adults and aged animals, as indicated by the vertical line on the right-hand side of the gel. d) Untreated or alkali-treated DNA from skeletal muscle of aged wt and SAMHD1 −/− (ko) mice was analyzed on a denaturing gel, and mtDNA was visualized using a COX1 probe. Each sample lane corresponds to an individual mouse, and dotted lines represent the median. e) The median length of the untreated mtDNA in samples from Fig. 5d is indicated by a horizontal line. The two groups were compared using Welch’s t-test (ns, non-significant; n = 4). f) The length difference between untreated and alkali-treated mtDNAs shown in Fig. 5d was used to compute the number of rNMPs per single strand of mtDNA. The horizontal lines indicate the median. The p-value of the statistically significant difference between the two groups was calculated by Welch’s t-test; n = 4. g) Long-range PCR was performed on mtDNA isolated from the TA muscle of adult and aged wt or SAMHD1 −/− (ko) mice. FL, full-length product; the vertical line indicates the size range of mtDNA molecules with deletions. h) Kaplan–Meier survival curve for wt and SAMHD1 −/− (ko) mice. Comparison of the curves by the log-rank (Mantel–Cox) test confirmed no statistically significant difference between the genotypes. The sizes of the bands in the DNA ladder are indicated in kb. See also Fig. S5.
    Figure Legend Snippet: Deletion of SAMHD1 does not affect mtDNA stability. a) MtDNA copy number in the TA muscle of 5 or 6 wt (filled dots) and SAMHD1 −/− (open dots) 13-week-old (adult), 1-year-old (old adult), and 2-year-old (aged) animals was determined by qPCR and normalized to the value for adult wt mice. The mean for each group is indicated by a horizontal line. The p-values were calculated using Welch’s t-test; ns, non-significant. b) DNA isolated from embryos and from the TA muscle of pups, adults, 1-year-old (old) adults, or aged animals was linearized with SacI endonuclease and separated on a neutral gel. MtDNA was visualized as above. Full-length mtDNA is indicated (FL); the asterisk denotes a higher-migrating species resistant to cleavage. c) Long-range PCR to detect deletions in mtDNA from the TA muscle of wt mice of various ages. Full-length product is indicated (FL). Only minor species containing deletions are observed in the mtDNA from old adults and aged animals, as indicated by the vertical line on the right-hand side of the gel. d) Untreated or alkali-treated DNA from skeletal muscle of aged wt and SAMHD1 −/− (ko) mice was analyzed on a denaturing gel, and mtDNA was visualized using a COX1 probe. Each sample lane corresponds to an individual mouse, and dotted lines represent the median. e) The median length of the untreated mtDNA in samples from Fig. 5d is indicated by a horizontal line. The two groups were compared using Welch’s t-test (ns, non-significant; n = 4). f) The length difference between untreated and alkali-treated mtDNAs shown in Fig. 5d was used to compute the number of rNMPs per single strand of mtDNA. The horizontal lines indicate the median. The p-value of the statistically significant difference between the two groups was calculated by Welch’s t-test; n = 4. g) Long-range PCR was performed on mtDNA isolated from the TA muscle of adult and aged wt or SAMHD1 −/− (ko) mice. FL, full-length product; the vertical line indicates the size range of mtDNA molecules with deletions. h) Kaplan–Meier survival curve for wt and SAMHD1 −/− (ko) mice. Comparison of the curves by the log-rank (Mantel–Cox) test confirmed no statistically significant difference between the genotypes. The sizes of the bands in the DNA ladder are indicated in kb. See also Fig. S5.

    Techniques Used: Real-time Polymerase Chain Reaction, Mouse Assay, Isolation, Polymerase Chain Reaction

    32) Product Images from "Physiological Studies of Escherichia coli Strain MG1655: Growth Defects and Apparent Cross-Regulation of Gene Expression"

    Article Title: Physiological Studies of Escherichia coli Strain MG1655: Growth Defects and Apparent Cross-Regulation of Gene Expression

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.185.18.5611-5626.2003

    PCR amplification of fnr region from different MG1655 isolates. The fnr region was amplified from the CGSC isolate of MG1655 (CGSC 6300; lane 1) and the isolate obtained from M. Singer and C. Gross (NCM3430; lane 2) (see Materials and Methods). The sizes of the molecular standards in lane 3 are noted to the right. The genes deleted in the CGSC isolate (b1332 to b1344) are, respectively, ynaJ (open reading frame conserved in E. coli and Salmonella enterica ), uspE ( ydaA ), fnr (Crp family activator of anaerobic respiratory gene transcription), ogt ( O -6-alkylguanine-DNA/cysteine-protein methyltransferase), abgT ( ydaH ; p ), abgB ( ydaI ; p ), abgA ( ydaJ ; p ), abgR ( ydaK ; p ), ydaL (open reading frame conserved in enterobacteria), ydaM (open reading frame conserved in E. coli ), ydaN (open reading frame conserved in enterobacteria), dbpA (ATP-dependent RNA helicase), and ydaO (open reading frame conserved in enterobacteria). The deletion is flanked by tns5_4 (b1331), which codes for IS 5 transposase, and ydaP (b1345), a rac prophage which codes for a putative prophage integrase.
    Figure Legend Snippet: PCR amplification of fnr region from different MG1655 isolates. The fnr region was amplified from the CGSC isolate of MG1655 (CGSC 6300; lane 1) and the isolate obtained from M. Singer and C. Gross (NCM3430; lane 2) (see Materials and Methods). The sizes of the molecular standards in lane 3 are noted to the right. The genes deleted in the CGSC isolate (b1332 to b1344) are, respectively, ynaJ (open reading frame conserved in E. coli and Salmonella enterica ), uspE ( ydaA ), fnr (Crp family activator of anaerobic respiratory gene transcription), ogt ( O -6-alkylguanine-DNA/cysteine-protein methyltransferase), abgT ( ydaH ; p ), abgB ( ydaI ; p ), abgA ( ydaJ ; p ), abgR ( ydaK ; p ), ydaL (open reading frame conserved in enterobacteria), ydaM (open reading frame conserved in E. coli ), ydaN (open reading frame conserved in enterobacteria), dbpA (ATP-dependent RNA helicase), and ydaO (open reading frame conserved in enterobacteria). The deletion is flanked by tns5_4 (b1331), which codes for IS 5 transposase, and ydaP (b1345), a rac prophage which codes for a putative prophage integrase.

    Techniques Used: Polymerase Chain Reaction, Amplification

    33) Product Images from "B-cell display-based one-step method to generate chimeric human IgG monoclonal antibodies"

    Article Title: B-cell display-based one-step method to generate chimeric human IgG monoclonal antibodies

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1122

    The chicken/human chimeric IgG expression construct. ( A ) Strategy for the knock-in of the human IgHG1 constant region at the chicken IgHM locus. The chicken IgHM region from the exon C H 1 to C H 3 was cloned to make the targeting vector. The intronic region digested by BseRI was replaced by the human IgHG1 region from H to C H 3 and a selectable marker. Bsd = blasticidin S deaminase. p(A) = polyadenylation sites. Genomic size and distances are not to scale (distance of chicken C H 1 to C H 2 = 4.5 kb; human H-C H 2-C H 3 insert = 1 kb). ( B ) Final genomic structure after integration of the knock-in plasmid and expected mRNA variants produced by alternative splicing. Thick horizontal bars indicate the approximate positions of the primers for PCR and RT–PCR used to confirm genomic integration and expression of IgHM and IgHG1 transcripts. ( C ) Expression of IgHM and of chimeric IgHG1 detected by RT–PCR in the wild-type parental strain (WT) and a positive chimeric transformant (CX13). ( D ) Schematic outline of the ADLib system applied to the selection of chimeric human IgG. (From left to right) The enhancement of sequence diversification at the Ig variable locus in TSA-treated DT40 cultures allows the generation of an autonomously diversifying library of cells expressing various surface IgM; the clones specific for the target antigen are isolated using antigen-coated magnetic beads; CX13 cells co-express secreted-form chicken IgM and chimeric IgG with the same antigen-binding domain; antigen-specific chimeric IgG can therefore be isolated directly from the culture supernatant for further use.
    Figure Legend Snippet: The chicken/human chimeric IgG expression construct. ( A ) Strategy for the knock-in of the human IgHG1 constant region at the chicken IgHM locus. The chicken IgHM region from the exon C H 1 to C H 3 was cloned to make the targeting vector. The intronic region digested by BseRI was replaced by the human IgHG1 region from H to C H 3 and a selectable marker. Bsd = blasticidin S deaminase. p(A) = polyadenylation sites. Genomic size and distances are not to scale (distance of chicken C H 1 to C H 2 = 4.5 kb; human H-C H 2-C H 3 insert = 1 kb). ( B ) Final genomic structure after integration of the knock-in plasmid and expected mRNA variants produced by alternative splicing. Thick horizontal bars indicate the approximate positions of the primers for PCR and RT–PCR used to confirm genomic integration and expression of IgHM and IgHG1 transcripts. ( C ) Expression of IgHM and of chimeric IgHG1 detected by RT–PCR in the wild-type parental strain (WT) and a positive chimeric transformant (CX13). ( D ) Schematic outline of the ADLib system applied to the selection of chimeric human IgG. (From left to right) The enhancement of sequence diversification at the Ig variable locus in TSA-treated DT40 cultures allows the generation of an autonomously diversifying library of cells expressing various surface IgM; the clones specific for the target antigen are isolated using antigen-coated magnetic beads; CX13 cells co-express secreted-form chicken IgM and chimeric IgG with the same antigen-binding domain; antigen-specific chimeric IgG can therefore be isolated directly from the culture supernatant for further use.

    Techniques Used: Expressing, Construct, Knock-In, Clone Assay, Plasmid Preparation, Marker, Produced, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Selection, Sequencing, Isolation, Magnetic Beads, Binding Assay

    34) Product Images from "An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers"

    Article Title: An efficient method for generation of bi-allelic null mutant mouse embryonic stem cells and its application for investigating epigenetic modifiers

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx811

    Generation and functional analysis of inducible conditional Setdb1 mutant ES cell lines. ( A ) Allelic structures and workflow of vector electroporations. IKMC heterozygous ‘knockout-first’ ( tm1a/ + ) ES cells are treated with Flp recombinase to generate a conditional allele ( tm1c ) and targeted with pI_hygGFP to generate conditional/null ( tm1c/tm2 ) cells. Cre-ERT2 is then introduced by targeting vector knockin into the Rosa26 locus. Upon treatment with 4′OHT, Cre recombinase activity removes the floxed ‘criticial exon’ of the tm1c allele to generate bi-allelic null cells ( tm1d/tm2 ). Primers for PCR genotyping are noted as small arrows. ( B ) Western blots showing absence of full-length Setdb1 protein (black arrow) in the Setdb1 tm1d/tm2 mutant ES cell lines after treatment with 4′OHT. A 39 kDa truncation product is generated from the tm2 allele (gray arrow). α-tubulin was used as a loading control (open arrow). ( C ) Setdb1 tm1d/tm2 mutant ES cells plated at low density (1 × 10∧3 cells per 10cm dish; following a 48 h 4′OHT treatment period) are unable to form colonies of undifferentiated ES cells, whereas control Setdb1 +/tm1d heterozygous ES cells (4′OHT treated and plated concurrently) exhibit normal undifferentiated ES cell colony morphology; a representative example is shown. Cells were stained with methylene blue 8 days after plating. ( D ) Growth of 4′OHT treated Setdb1 tm1d/tm2 null ES cells is severely compromised, while 4′OHT treated control Setdb1 +/tm1d heterozygous ES cells retain robust growth characteristics similar to non-treated Setdb1 tm1c/ + and tm1c/tm2 cells. Cells were plated following a 48 h 4′OHT treatment and counted at the time intervals indicated. Data points are the mean of three biological replicates (independent cell lines), error bars indicate s.d. ( E ) 4′OHT-treated Setdb1 tm1c/tm2 ES cells start to differentiate by 6 days after treatment and gradually lose alkaline phosphatase (AP) activity, while treated control Setdb1 +/tm1d ES cells retain AP activity and normal ES cell morphology.
    Figure Legend Snippet: Generation and functional analysis of inducible conditional Setdb1 mutant ES cell lines. ( A ) Allelic structures and workflow of vector electroporations. IKMC heterozygous ‘knockout-first’ ( tm1a/ + ) ES cells are treated with Flp recombinase to generate a conditional allele ( tm1c ) and targeted with pI_hygGFP to generate conditional/null ( tm1c/tm2 ) cells. Cre-ERT2 is then introduced by targeting vector knockin into the Rosa26 locus. Upon treatment with 4′OHT, Cre recombinase activity removes the floxed ‘criticial exon’ of the tm1c allele to generate bi-allelic null cells ( tm1d/tm2 ). Primers for PCR genotyping are noted as small arrows. ( B ) Western blots showing absence of full-length Setdb1 protein (black arrow) in the Setdb1 tm1d/tm2 mutant ES cell lines after treatment with 4′OHT. A 39 kDa truncation product is generated from the tm2 allele (gray arrow). α-tubulin was used as a loading control (open arrow). ( C ) Setdb1 tm1d/tm2 mutant ES cells plated at low density (1 × 10∧3 cells per 10cm dish; following a 48 h 4′OHT treatment period) are unable to form colonies of undifferentiated ES cells, whereas control Setdb1 +/tm1d heterozygous ES cells (4′OHT treated and plated concurrently) exhibit normal undifferentiated ES cell colony morphology; a representative example is shown. Cells were stained with methylene blue 8 days after plating. ( D ) Growth of 4′OHT treated Setdb1 tm1d/tm2 null ES cells is severely compromised, while 4′OHT treated control Setdb1 +/tm1d heterozygous ES cells retain robust growth characteristics similar to non-treated Setdb1 tm1c/ + and tm1c/tm2 cells. Cells were plated following a 48 h 4′OHT treatment and counted at the time intervals indicated. Data points are the mean of three biological replicates (independent cell lines), error bars indicate s.d. ( E ) 4′OHT-treated Setdb1 tm1c/tm2 ES cells start to differentiate by 6 days after treatment and gradually lose alkaline phosphatase (AP) activity, while treated control Setdb1 +/tm1d ES cells retain AP activity and normal ES cell morphology.

    Techniques Used: Functional Assay, Mutagenesis, Plasmid Preparation, Knock-Out, Knock-In, Activity Assay, Polymerase Chain Reaction, Western Blot, Generated, Staining

    Schematic of allele structures in second allele targeted and revertant ES cells and cell line validation. ( A ) Insertion-type targeting vector pI_hygGFP for inactivation of the WT allele in ES cells heterozygous for a standard IKMC knockout-first allele ( tm1a ). ( B ) Structure of the bi-allelic locus after targeting the second allele. The tm2 allele contains the pI_hygGFP targeting cassette and duplicated homology region, where exon 2 is re-generated by gap repair. ( C ) Western blots of Cbx1 and Jarid2 parental IKMC heterozygous ES cells (B01 and E08 lines), and examples of cell lines following pI_hygGFP electroporation including doubly targeted ES cells ( 1a/2 ) showing the absence of protein expression, and failed targeting events ( 1a/+ ). ( D ) Reversion from null mutant ( 1a/2 ) to conditional mutant ( 1c/2 ) by Flp recombinase. ( E ) Western blots of mutant ( 1a/2 ) and reverted ( 1c/2 ) Cbx1 and Jarid2 ES cell lines showing re-expression of protein. Primers for LR-PCR genotyping are indicated by small arrows and α-tubulin was used for Western blot loading controls. ( F ) Rescue of Polycomb PRC1 recruitment to PRC2 target genes in Jarid2 revertant cell lines ( 1c/2 ), shown by reinstatement of Mel18 binding at known Jarid2-dependent gene promoter regions, assessed by chromatin immunoprecipitation (ChIP)-qRT-PCR. Hprt is a control locus known to be negative for PRC1 binding. Results show mean ± s.d. of three biological replicates (independent cell lines), where values are expressed as relative fold-enrichment over 10% input chromatin. Asterisks indicate statistically significant differences between Jarid2 revertant ( 1c/2 ) and null ( 1a/2 ) cell lines ( P
    Figure Legend Snippet: Schematic of allele structures in second allele targeted and revertant ES cells and cell line validation. ( A ) Insertion-type targeting vector pI_hygGFP for inactivation of the WT allele in ES cells heterozygous for a standard IKMC knockout-first allele ( tm1a ). ( B ) Structure of the bi-allelic locus after targeting the second allele. The tm2 allele contains the pI_hygGFP targeting cassette and duplicated homology region, where exon 2 is re-generated by gap repair. ( C ) Western blots of Cbx1 and Jarid2 parental IKMC heterozygous ES cells (B01 and E08 lines), and examples of cell lines following pI_hygGFP electroporation including doubly targeted ES cells ( 1a/2 ) showing the absence of protein expression, and failed targeting events ( 1a/+ ). ( D ) Reversion from null mutant ( 1a/2 ) to conditional mutant ( 1c/2 ) by Flp recombinase. ( E ) Western blots of mutant ( 1a/2 ) and reverted ( 1c/2 ) Cbx1 and Jarid2 ES cell lines showing re-expression of protein. Primers for LR-PCR genotyping are indicated by small arrows and α-tubulin was used for Western blot loading controls. ( F ) Rescue of Polycomb PRC1 recruitment to PRC2 target genes in Jarid2 revertant cell lines ( 1c/2 ), shown by reinstatement of Mel18 binding at known Jarid2-dependent gene promoter regions, assessed by chromatin immunoprecipitation (ChIP)-qRT-PCR. Hprt is a control locus known to be negative for PRC1 binding. Results show mean ± s.d. of three biological replicates (independent cell lines), where values are expressed as relative fold-enrichment over 10% input chromatin. Asterisks indicate statistically significant differences between Jarid2 revertant ( 1c/2 ) and null ( 1a/2 ) cell lines ( P

    Techniques Used: Plasmid Preparation, Knock-Out, Generated, Western Blot, Electroporation, Expressing, Mutagenesis, Polymerase Chain Reaction, Binding Assay, Chromatin Immunoprecipitation, Quantitative RT-PCR

    35) Product Images from "Structural polymorphism in the promoter of pfmrp2 confers Plasmodium falciparum tolerance to quinoline drugs"

    Article Title: Structural polymorphism in the promoter of pfmrp2 confers Plasmodium falciparum tolerance to quinoline drugs

    Journal: Molecular Microbiology

    doi: 10.1111/mmi.12505

    Deletion of the pfmrp2 gene 5′ upstream sequence identified by PCR and sequencing.A. A map of the 5′ upstream region of the 3D7 pfmrp2 gene showing the panel of used primer pairs (purple) and their positions relative to the gene start codon. Listed are the sizes of the PCR products obtained for clones 11C/wt and 6A/mut for the various primer pairs. Bottom panel: Agarose gel images of PCR products obtained for the corresponding primer pairs. The images show full-length PCR products obtained for clone 11C/wt for all primer pairs and the truncated products for the primer pairs 1.9 and 1.12 from 6A/mut.B. Sequence alignment of clone 11C/wt and clone 6A/mut compared to 3D7 sequence (PlasmoDB version 8.1). The sequence alignment depicts the 200 bp coding sequence of the pfmrp2 gene at the 5′ end and its upstream, non-coding region. The grey dotted lines indicate the break points of the 6A/mut deletion. The numbers indicate the base position relative to the start codon. Two DNA constructs of 6A/mut of 0.3 and 0.5 kb in size were sequenced to identify the deletion break point as −4345th bp from the start codon.
    Figure Legend Snippet: Deletion of the pfmrp2 gene 5′ upstream sequence identified by PCR and sequencing.A. A map of the 5′ upstream region of the 3D7 pfmrp2 gene showing the panel of used primer pairs (purple) and their positions relative to the gene start codon. Listed are the sizes of the PCR products obtained for clones 11C/wt and 6A/mut for the various primer pairs. Bottom panel: Agarose gel images of PCR products obtained for the corresponding primer pairs. The images show full-length PCR products obtained for clone 11C/wt for all primer pairs and the truncated products for the primer pairs 1.9 and 1.12 from 6A/mut.B. Sequence alignment of clone 11C/wt and clone 6A/mut compared to 3D7 sequence (PlasmoDB version 8.1). The sequence alignment depicts the 200 bp coding sequence of the pfmrp2 gene at the 5′ end and its upstream, non-coding region. The grey dotted lines indicate the break points of the 6A/mut deletion. The numbers indicate the base position relative to the start codon. Two DNA constructs of 6A/mut of 0.3 and 0.5 kb in size were sequenced to identify the deletion break point as −4345th bp from the start codon.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Clone Assay, Agarose Gel Electrophoresis, Construct

    36) Product Images from "Correction of the auditory phenotype in C57BL/6N mice via CRISPR/Cas9-mediated homology directed repair"

    Article Title: Correction of the auditory phenotype in C57BL/6N mice via CRISPR/Cas9-mediated homology directed repair

    Journal: Genome Medicine

    doi: 10.1186/s13073-016-0273-4

    Design of two targeting strategies to recover normal splicing/function of the C57BL/6NTac Cdh23 gene. a Design 1 utilises a 121 bp single-stranded oligonucleotide donor ( ssODN_U1 ) in combination with two single guide RNAs ( sgRNA_U1 and sgRNA_D1 ), which flank the Cdh23 ahl allele. The full ssODN_U1 donor sequence is shown below the C57BL/6NTac Cdh23 ahl sequence ( uppercase denotes exonic sequence and lowercase denotes intronic sequence), with homology indicated by dashed lines , the Cdh23 ahl allele with an arrow , and two base changes shown in red text . The two synonymous base substitutions are designed to prevent further modification by the CRISPR/Cas9 following repair. The corrected Cdh23 753A > G allele is the allele found in inbred mouse strains that do not demonstrate age-related hearing loss (ARHL). The final corrected Cdh23 753A > G(U1) gene sequence closely matches that found in these non-ARHL inbred strains at the nucleotide level, except for the two synonymous base substitutions (c.724A > T and c.725G > C; red text ). The Cdh23 753A > G(U1) product is predicted to be identical to the Cdh23 protein found in non-ARHL mouse strains. b Design 2 also utilises a 121 bp ssODN ( ssODN_U2 ) in combination with two single guide RNAs, sgRNA_D1 also used in design 1 and sgRNA_U2, which lies across the Cdh23 ahl locus. The full ssODN_U2 donor sequence is shown below the C57BL/6NTac Cdh23 ahl sequence, with homology indicated by dashed lines , the Cdh23 ahl allele with an arrow , and one intronic base change shown in red text . The intronic base substitution is designed to prevent further modification by the CRISPR/Cas9 following repair. The final corrected Cdh23 753A > G(U2) gene sequence is identical to that found in non-ARHL inbred strains at the nucleotide level, with only an intronic base substitution (c.753 + 9c > t; red text ). The Cdh23 753A > G(U2) product is predicted to be identical to the Cdh23 protein found in non-ARHL mouse strains
    Figure Legend Snippet: Design of two targeting strategies to recover normal splicing/function of the C57BL/6NTac Cdh23 gene. a Design 1 utilises a 121 bp single-stranded oligonucleotide donor ( ssODN_U1 ) in combination with two single guide RNAs ( sgRNA_U1 and sgRNA_D1 ), which flank the Cdh23 ahl allele. The full ssODN_U1 donor sequence is shown below the C57BL/6NTac Cdh23 ahl sequence ( uppercase denotes exonic sequence and lowercase denotes intronic sequence), with homology indicated by dashed lines , the Cdh23 ahl allele with an arrow , and two base changes shown in red text . The two synonymous base substitutions are designed to prevent further modification by the CRISPR/Cas9 following repair. The corrected Cdh23 753A > G allele is the allele found in inbred mouse strains that do not demonstrate age-related hearing loss (ARHL). The final corrected Cdh23 753A > G(U1) gene sequence closely matches that found in these non-ARHL inbred strains at the nucleotide level, except for the two synonymous base substitutions (c.724A > T and c.725G > C; red text ). The Cdh23 753A > G(U1) product is predicted to be identical to the Cdh23 protein found in non-ARHL mouse strains. b Design 2 also utilises a 121 bp ssODN ( ssODN_U2 ) in combination with two single guide RNAs, sgRNA_D1 also used in design 1 and sgRNA_U2, which lies across the Cdh23 ahl locus. The full ssODN_U2 donor sequence is shown below the C57BL/6NTac Cdh23 ahl sequence, with homology indicated by dashed lines , the Cdh23 ahl allele with an arrow , and one intronic base change shown in red text . The intronic base substitution is designed to prevent further modification by the CRISPR/Cas9 following repair. The final corrected Cdh23 753A > G(U2) gene sequence is identical to that found in non-ARHL inbred strains at the nucleotide level, with only an intronic base substitution (c.753 + 9c > t; red text ). The Cdh23 753A > G(U2) product is predicted to be identical to the Cdh23 protein found in non-ARHL mouse strains

    Techniques Used: Sequencing, Modification, CRISPR

    37) Product Images from "Chemoresistance to Valproate Treatment of Bovine Leukemia Virus-Infected Sheep; Identification of Improved HDAC Inhibitors"

    Article Title: Chemoresistance to Valproate Treatment of Bovine Leukemia Virus-Infected Sheep; Identification of Improved HDAC Inhibitors

    Journal: Pathogens

    doi: 10.3390/pathogens1020065

    BLV proviral integrity and integration sites during VPA treatment and relapse: A. DNA was extracted from BLV-infected sheep PBMCs (2213, 3002, 3003, 4213, 4535) isolated just before VPA treatment at day 0 (B) and at the end of the relapse period (R). The full length proviral sequence was amplified by PCR using primers located in the 5' and 3' LTRs. PCR amplification products were migrated onto an agarose gel. The molecular weight of the amplicon is indicated in kilobases (8 Kb). B. Southern blot hybridization using a BLV probe of genomic DNA digested with EcoRI. The molecular weight markers (in Kb) are indicated on the left. Lanes correspond to those of panel A.
    Figure Legend Snippet: BLV proviral integrity and integration sites during VPA treatment and relapse: A. DNA was extracted from BLV-infected sheep PBMCs (2213, 3002, 3003, 4213, 4535) isolated just before VPA treatment at day 0 (B) and at the end of the relapse period (R). The full length proviral sequence was amplified by PCR using primers located in the 5' and 3' LTRs. PCR amplification products were migrated onto an agarose gel. The molecular weight of the amplicon is indicated in kilobases (8 Kb). B. Southern blot hybridization using a BLV probe of genomic DNA digested with EcoRI. The molecular weight markers (in Kb) are indicated on the left. Lanes correspond to those of panel A.

    Techniques Used: Infection, Isolation, Sequencing, Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Molecular Weight, Southern Blot, Hybridization

    BLV proviral loads during VPA treatment and relapse: The proviral loads (empty squares), represented as numbers of viral copies per 10 3 B-lymphocytes, were measured by real time PCR using genomic DNA isolated from sheep peripheral blood mononuclear cells (PBMCs). Data result from triplicate measurements (error bars represent means ± standard deviations). Absolute leukocyte numbers are indicated as reference (dotted line).
    Figure Legend Snippet: BLV proviral loads during VPA treatment and relapse: The proviral loads (empty squares), represented as numbers of viral copies per 10 3 B-lymphocytes, were measured by real time PCR using genomic DNA isolated from sheep peripheral blood mononuclear cells (PBMCs). Data result from triplicate measurements (error bars represent means ± standard deviations). Absolute leukocyte numbers are indicated as reference (dotted line).

    Techniques Used: Real-time Polymerase Chain Reaction, Isolation

    38) Product Images from "Widespread somatic L1 retrotransposition occurs early during gastrointestinal cancer evolution"

    Article Title: Widespread somatic L1 retrotransposition occurs early during gastrointestinal cancer evolution

    Journal: Genome Research

    doi: 10.1101/gr.196238.115

    Representative examples of further somatic L1 insertions in colon cancer patient 2BV and gastric cancer patient 2043. ( A ) L1 insertions in colorectal cancer (all detectable by conventional PCR), from left to right : ins. D2 (polyp-specific, present exclusively
    Figure Legend Snippet: Representative examples of further somatic L1 insertions in colon cancer patient 2BV and gastric cancer patient 2043. ( A ) L1 insertions in colorectal cancer (all detectable by conventional PCR), from left to right : ins. D2 (polyp-specific, present exclusively

    Techniques Used: Polymerase Chain Reaction

    Timing and distribution of somatic L1 insertions in GI cancers
    Figure Legend Snippet: Timing and distribution of somatic L1 insertions in GI cancers

    Techniques Used:

    Genomic distribution of L1 insertions in GI tumors. ( A ) The genomic distribution of reference (light blue histogram) (Supplemental Table 3) and putative nonreference L1 insertions (light red histogram) (Supplemental Table 4) in colon cancer cases is shown
    Figure Legend Snippet: Genomic distribution of L1 insertions in GI tumors. ( A ) The genomic distribution of reference (light blue histogram) (Supplemental Table 3) and putative nonreference L1 insertions (light red histogram) (Supplemental Table 4) in colon cancer cases is shown

    Techniques Used:

    Distribution of somatic L1 insertions. ( A ) Normal colon crypt containing a few cells with L1 insertions (detectable only by nested PCR). ( B ) Colon tumor with early L1 insertions (detectable by conventional PCR). ( C ) L1 insertions occurring late during
    Figure Legend Snippet: Distribution of somatic L1 insertions. ( A ) Normal colon crypt containing a few cells with L1 insertions (detectable only by nested PCR). ( B ) Colon tumor with early L1 insertions (detectable by conventional PCR). ( C ) L1 insertions occurring late during

    Techniques Used: Nested PCR, Polymerase Chain Reaction

    39) Product Images from "Epidermal growth factor receptor variant III mutations in lung tumorigenesis and sensitivity to tyrosine kinase inhibitors"

    Article Title: Epidermal growth factor receptor variant III mutations in lung tumorigenesis and sensitivity to tyrosine kinase inhibitors

    Journal:

    doi: 10.1073/pnas.0510284103

    Immunostaining analyses of the lung SCC tumor samples positive for EGFRvIII. Immunostaining of EGFRvIII-specific antibody DH8.3 was preformed as described in Materials and Methods . ( A ) Glioblastoma sample positive for EGFRvIII from both RT-PCR and FISH
    Figure Legend Snippet: Immunostaining analyses of the lung SCC tumor samples positive for EGFRvIII. Immunostaining of EGFRvIII-specific antibody DH8.3 was preformed as described in Materials and Methods . ( A ) Glioblastoma sample positive for EGFRvIII from both RT-PCR and FISH

    Techniques Used: Immunostaining, Reverse Transcription Polymerase Chain Reaction, Fluorescence In Situ Hybridization

    HKI-272 is effective in growth inhibition of Ba/F3 cells transformed with EGFRvIII, EGFR-L858R, and EGFR-L858R-T790M but fail to inhibit the Ba/F3 cells transformed with EGFRvIIIC530S. The sensitivity of various EGFR mutant-transformed Ba/F3 cells to
    Figure Legend Snippet: HKI-272 is effective in growth inhibition of Ba/F3 cells transformed with EGFRvIII, EGFR-L858R, and EGFR-L858R-T790M but fail to inhibit the Ba/F3 cells transformed with EGFRvIIIC530S. The sensitivity of various EGFR mutant-transformed Ba/F3 cells to

    Techniques Used: Inhibition, Transformation Assay, Mutagenesis

    Identification of EGFRvIII in human NSCLC at both RNA and genomic DNA levels. ( A Left ) Detection of WT EGFR (929 bp) and EGFRvIII (128 bp) in NSCLC by RT-PCR. Lanes are as follows: M, marker; 1, 97-19-tumor; 2, 4040-tumor; 3, 4050088A2-tumor; 4, 54943-tumor;
    Figure Legend Snippet: Identification of EGFRvIII in human NSCLC at both RNA and genomic DNA levels. ( A Left ) Detection of WT EGFR (929 bp) and EGFRvIII (128 bp) in NSCLC by RT-PCR. Lanes are as follows: M, marker; 1, 97-19-tumor; 2, 4040-tumor; 3, 4050088A2-tumor; 4, 54943-tumor;

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Marker

    Treatment of the lung tumors in the Tet-op-EGFRvIII/CCSP-rtTA, Ink4A/Arf −/− mice with EGFR inhibitors. The Tet-op-EGFRvIII/CCSP-rtTA, Ink4A/Arf −/− mice given doxycycline water for > 8 weeks were imaged with MRI (see
    Figure Legend Snippet: Treatment of the lung tumors in the Tet-op-EGFRvIII/CCSP-rtTA, Ink4A/Arf −/− mice with EGFR inhibitors. The Tet-op-EGFRvIII/CCSP-rtTA, Ink4A/Arf −/− mice given doxycycline water for > 8 weeks were imaged with MRI (see

    Techniques Used: Mouse Assay, Magnetic Resonance Imaging

    Expression of EGFRvIII in mouse lungs leads to the development of lung cancer in vivo . ( A ) Monotransgenic Tet-op-EGFRvIII mice had normal lungs with no lesions observable after 4 months of doxycycline administration. ( B and C ) Bitransgenic mice and Tet-op-EGFRvIII/CCSP-rtTA
    Figure Legend Snippet: Expression of EGFRvIII in mouse lungs leads to the development of lung cancer in vivo . ( A ) Monotransgenic Tet-op-EGFRvIII mice had normal lungs with no lesions observable after 4 months of doxycycline administration. ( B and C ) Bitransgenic mice and Tet-op-EGFRvIII/CCSP-rtTA

    Techniques Used: Expressing, In Vivo, Mouse Assay

    40) Product Images from "Genetically enhanced asynapsis of autosomal chromatin promotes transcriptional dysregulation and meiotic failure"

    Article Title: Genetically enhanced asynapsis of autosomal chromatin promotes transcriptional dysregulation and meiotic failure

    Journal: Chromosoma

    doi: 10.1007/s00412-011-0346-5

    Tcp1 , Tsga2 , and Scml2 expression by RNA FISH of control and T43H pachytene spermatocytes. a Expression of Tcp1 ( green ), (chr17:13,109,331–13,117,933) in late zygotene–early pachytene ( upper panel ), and in mid–late pachytene cells ( lower panel ) of B6 and t 12 /B10 fertile males and T+/+ T43H ( T /T43H) and t 12 +/+ T43H ( t 12 /T43H) sterile males. b Expression of Tsga2 (chr17:31,391,965–31,414,301) ( green ). c Expression of Scml2 (chrX:157,555,125–157,696,145) ( green ). The substages of zygotene–pachytene and mid–late pachytene spermatocytes were discriminated by pattern of γH2AX immunostaining ( red ). At transition of late zygonema to early leptonema, the γH2AX disappears from autosomal bivalents and simultaneously elevates its content on the condensing sex chromatin; in mid–late pachynema, γH2AX is associated with the condensed chromatin of the sex body. However, it is completely absent on autosomes except of unsynapsed translocation quadrivalent in sterile males. The chromatin is stained by DAPI ( blue ). Positive RNA FISH signals are marked by arrows
    Figure Legend Snippet: Tcp1 , Tsga2 , and Scml2 expression by RNA FISH of control and T43H pachytene spermatocytes. a Expression of Tcp1 ( green ), (chr17:13,109,331–13,117,933) in late zygotene–early pachytene ( upper panel ), and in mid–late pachytene cells ( lower panel ) of B6 and t 12 /B10 fertile males and T+/+ T43H ( T /T43H) and t 12 +/+ T43H ( t 12 /T43H) sterile males. b Expression of Tsga2 (chr17:31,391,965–31,414,301) ( green ). c Expression of Scml2 (chrX:157,555,125–157,696,145) ( green ). The substages of zygotene–pachytene and mid–late pachytene spermatocytes were discriminated by pattern of γH2AX immunostaining ( red ). At transition of late zygonema to early leptonema, the γH2AX disappears from autosomal bivalents and simultaneously elevates its content on the condensing sex chromatin; in mid–late pachynema, γH2AX is associated with the condensed chromatin of the sex body. However, it is completely absent on autosomes except of unsynapsed translocation quadrivalent in sterile males. The chromatin is stained by DAPI ( blue ). Positive RNA FISH signals are marked by arrows

    Techniques Used: Expressing, Fluorescence In Situ Hybridization, Immunostaining, Translocation Assay, Staining

    Related Articles

    Real-time Polymerase Chain Reaction:

    Article Title: Dynamic Distribution of Linker Histone H1.5 in Cellular Differentiation
    Article Snippet: .. ChIP–quantitative PCR Real-time PCR was performed on ChIP and input DNA using SYBR Green Real-time PCR Master Mix (Roche). .. For each primer pair, an amplification standard curve was established by gradient amount of input DNA.

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation
    Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) Total RNA was isolated from two million cells by SV total RNA isolation kit (Promega). cDNA was prepared by annealing 500 ng RNA with oligo dT as per the manufacturer’s instructions (Transcriptor High Fidelity cDNA Synthesis kit, Roche). ..

    Transfection:

    Article Title: Hepatitis B Viral DNA Decline at Loss of HBeAg Is Mainly Explained by Reduced cccDNA Load - Down-Regulated Transcription of PgRNA Has Limited Impact
    Article Snippet: .. Extraction of DNA and RNA from Cells DNA and RNA were extracted from transfected and non-transfected hepatoma cells in a Magnapure robot (Roche Applied Science, Germany) using the Total NA protocol. .. Harvested cells were washed in 1 mL PBS and after centrifugation at 5000 rpm for 3 min the pellet was re-suspended in 800 µL RLT lysis buffer (Qiagen Sciences, MD, USA) before extraction.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1, et al. Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1
    Article Snippet: .. 2.5 Real‐time quantitative RT‐PCR First‐strand cDNA was synthesized from the total RNA using ThermoScript RT‐PCR System (Roche, Indianapolis, IN, USA). .. PCR was performed on a LightCycler system (Roche) using LightCycler FastStart DNA Master SYBR Green I reaction mix (Roche) and QuantiTect Primer Assays (QIAGEN, Hilden, Germany).

    Synthesized:

    Article Title: Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1, et al. Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1
    Article Snippet: .. 2.5 Real‐time quantitative RT‐PCR First‐strand cDNA was synthesized from the total RNA using ThermoScript RT‐PCR System (Roche, Indianapolis, IN, USA). .. PCR was performed on a LightCycler system (Roche) using LightCycler FastStart DNA Master SYBR Green I reaction mix (Roche) and QuantiTect Primer Assays (QIAGEN, Hilden, Germany).

    Isolation:

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation
    Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) Total RNA was isolated from two million cells by SV total RNA isolation kit (Promega). cDNA was prepared by annealing 500 ng RNA with oligo dT as per the manufacturer’s instructions (Transcriptor High Fidelity cDNA Synthesis kit, Roche). ..

    Functional Assay:

    Article Title: Mitochondrial genome evolution in species belonging to the Phialocephala fortinii s.l. - Acephala applanata species complex
    Article Snippet: .. Sequencing the complete mt genome of Phialocephala subalpina In the course of a genome sequencing project of P. subalpina strain UAMH 11012, an initial Roche/454 GS FLX (454) shotgun run was performed at the Functional Genomics Centre Zurich (FGCZ, Uni/ETH Zurich) and from that a draft of the circular mt genome of P. subalpina strain UAMH 11012 became available. .. The draft sequence was subdivided into 12 fragments (see Additional file ) and amplified from strain UAMH 11012 using long-range PCR in 20 μl volumes (Expand Long Range dNTPack kit, Roche, Rotkreuz, Switzerland).

    Quantitative RT-PCR:

    Article Title: Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1, et al. Chronic hypoxia‐induced slug promotes invasive behavior of prostate cancer cells by activating expression of ephrin‐B1
    Article Snippet: .. 2.5 Real‐time quantitative RT‐PCR First‐strand cDNA was synthesized from the total RNA using ThermoScript RT‐PCR System (Roche, Indianapolis, IN, USA). .. PCR was performed on a LightCycler system (Roche) using LightCycler FastStart DNA Master SYBR Green I reaction mix (Roche) and QuantiTect Primer Assays (QIAGEN, Hilden, Germany).

    Article Title: Genome-wide analyses reveal the IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation
    Article Snippet: .. Reverse transcription quantitative PCR (RT-qPCR) Total RNA was isolated from two million cells by SV total RNA isolation kit (Promega). cDNA was prepared by annealing 500 ng RNA with oligo dT as per the manufacturer’s instructions (Transcriptor High Fidelity cDNA Synthesis kit, Roche). ..

    SYBR Green Assay:

    Article Title: Dynamic Distribution of Linker Histone H1.5 in Cellular Differentiation
    Article Snippet: .. ChIP–quantitative PCR Real-time PCR was performed on ChIP and input DNA using SYBR Green Real-time PCR Master Mix (Roche). .. For each primer pair, an amplification standard curve was established by gradient amount of input DNA.

    Polymerase Chain Reaction:

    Article Title: Dynamic Distribution of Linker Histone H1.5 in Cellular Differentiation
    Article Snippet: .. ChIP–quantitative PCR Real-time PCR was performed on ChIP and input DNA using SYBR Green Real-time PCR Master Mix (Roche). .. For each primer pair, an amplification standard curve was established by gradient amount of input DNA.

    Sequencing:

    Article Title: Mitochondrial genome evolution in species belonging to the Phialocephala fortinii s.l. - Acephala applanata species complex
    Article Snippet: .. Sequencing the complete mt genome of Phialocephala subalpina In the course of a genome sequencing project of P. subalpina strain UAMH 11012, an initial Roche/454 GS FLX (454) shotgun run was performed at the Functional Genomics Centre Zurich (FGCZ, Uni/ETH Zurich) and from that a draft of the circular mt genome of P. subalpina strain UAMH 11012 became available. .. The draft sequence was subdivided into 12 fragments (see Additional file ) and amplified from strain UAMH 11012 using long-range PCR in 20 μl volumes (Expand Long Range dNTPack kit, Roche, Rotkreuz, Switzerland).

    Chromatin Immunoprecipitation:

    Article Title: Dynamic Distribution of Linker Histone H1.5 in Cellular Differentiation
    Article Snippet: .. ChIP–quantitative PCR Real-time PCR was performed on ChIP and input DNA using SYBR Green Real-time PCR Master Mix (Roche). .. For each primer pair, an amplification standard curve was established by gradient amount of input DNA.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92
    Roche long range pcr
    Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 1A/1B-2 by long-range <t>PCR</t> and sequencing of the breakpoints. (B) Confirmation of the deletion of exons 5–14 by long-range PCR and sequencing of the breakpoints. (C) Confirmation of the deletion of exons 11–12 by long-range PCR and sequencing of the breakpoints. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb <t>DNA</t> Ladder, Invitrogen).
    Long Range Pcr, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/long range pcr/product/Roche
    Average 92 stars, based on 170 article reviews
    Price from $9.99 to $1999.99
    long range pcr - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    94
    Roche expand long template pcr system
    Deletion of SAMHD1 does not affect <t>mtDNA</t> stability. a) MtDNA copy number in the TA muscle of 5 or 6 wt (filled dots) and SAMHD1 −/− (open dots) 13-week-old (adult), 1-year-old (old adult), and 2-year-old (aged) animals was determined by qPCR and normalized to the value for adult wt mice. The mean for each group is indicated by a horizontal line. The p-values were calculated using Welch’s t-test; ns, non-significant. b) DNA isolated from embryos and from the TA muscle of pups, adults, 1-year-old (old) adults, or aged animals was linearized with SacI endonuclease and separated on a neutral gel. MtDNA was visualized as above. Full-length mtDNA is indicated (FL); the asterisk denotes a higher-migrating species resistant to cleavage. c) Long-range <t>PCR</t> to detect deletions in mtDNA from the TA muscle of wt mice of various ages. Full-length product is indicated (FL). Only minor species containing deletions are observed in the mtDNA from old adults and aged animals, as indicated by the vertical line on the right-hand side of the gel. d) Untreated or alkali-treated DNA from skeletal muscle of aged wt and SAMHD1 −/− (ko) mice was analyzed on a denaturing gel, and mtDNA was visualized using a COX1 probe. Each sample lane corresponds to an individual mouse, and dotted lines represent the median. e) The median length of the untreated mtDNA in samples from Fig. 5d is indicated by a horizontal line. The two groups were compared using Welch’s t-test (ns, non-significant; n = 4). f) The length difference between untreated and alkali-treated mtDNAs shown in Fig. 5d was used to compute the number of rNMPs per single strand of mtDNA. The horizontal lines indicate the median. The p-value of the statistically significant difference between the two groups was calculated by Welch’s t-test; n = 4. g) Long-range PCR was performed on mtDNA isolated from the TA muscle of adult and aged wt or SAMHD1 −/− (ko) mice. FL, full-length product; the vertical line indicates the size range of mtDNA molecules with deletions. h) Kaplan–Meier survival curve for wt and SAMHD1 −/− (ko) mice. Comparison of the curves by the log-rank (Mantel–Cox) test confirmed no statistically significant difference between the genotypes. The sizes of the bands in the DNA ladder are indicated in kb. See also Fig. S5.
    Expand Long Template Pcr System, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 1004 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/expand long template pcr system/product/Roche
    Average 94 stars, based on 1004 article reviews
    Price from $9.99 to $1999.99
    expand long template pcr system - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    Image Search Results


    Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 1A/1B-2 by long-range PCR and sequencing of the breakpoints. (B) Confirmation of the deletion of exons 5–14 by long-range PCR and sequencing of the breakpoints. (C) Confirmation of the deletion of exons 11–12 by long-range PCR and sequencing of the breakpoints. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

    Journal: BMC Medical Genetics

    Article Title: High occurrence of BRCA1 intragenic rearrangements in hereditary breast and ovarian cancer syndrome in the Czech Republic

    doi: 10.1186/1471-2350-8-32

    Figure Lengend Snippet: Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 1A/1B-2 by long-range PCR and sequencing of the breakpoints. (B) Confirmation of the deletion of exons 5–14 by long-range PCR and sequencing of the breakpoints. (C) Confirmation of the deletion of exons 11–12 by long-range PCR and sequencing of the breakpoints. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

    Article Snippet: Confirmation and characterization of the rearrangements Positive results detected by MLPA of two independently drawn samples of genomic DNA were confirmed by long-range PCR (Expand Long Template PCR System, Roche Applied Science), conducted in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction, Sequencing, Negative Control, Marker

    Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 18–19 by long-range PCR and sequencing of the breakpoints. (B) Confirmation of the deletion of exon 20 and sequencing of the breakpoints.Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

    Journal: BMC Medical Genetics

    Article Title: High occurrence of BRCA1 intragenic rearrangements in hereditary breast and ovarian cancer syndrome in the Czech Republic

    doi: 10.1186/1471-2350-8-32

    Figure Lengend Snippet: Confirmation and characterization of the rearrangements . (A) Confirmation of the deletion of exons 18–19 by long-range PCR and sequencing of the breakpoints. (B) Confirmation of the deletion of exon 20 and sequencing of the breakpoints.Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

    Article Snippet: Confirmation and characterization of the rearrangements Positive results detected by MLPA of two independently drawn samples of genomic DNA were confirmed by long-range PCR (Expand Long Template PCR System, Roche Applied Science), conducted in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction, Sequencing, Negative Control, Marker

    Confirmation and characterization of the rearrangements . Confirmation of the deletion of the exons 21–22 by long-range PCR and sequencing of the breakpoints. The deletion/insertion event was characterized as g.77128_80906del3779ins236. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

    Journal: BMC Medical Genetics

    Article Title: High occurrence of BRCA1 intragenic rearrangements in hereditary breast and ovarian cancer syndrome in the Czech Republic

    doi: 10.1186/1471-2350-8-32

    Figure Lengend Snippet: Confirmation and characterization of the rearrangements . Confirmation of the deletion of the exons 21–22 by long-range PCR and sequencing of the breakpoints. The deletion/insertion event was characterized as g.77128_80906del3779ins236. Lanes 1+, 2+, carriers of the deletion; lane C-, negative control (wt); lane B, blank; lane M, marker (Ready-Load™ 1 Kb DNA Ladder, Invitrogen).

    Article Snippet: Confirmation and characterization of the rearrangements Positive results detected by MLPA of two independently drawn samples of genomic DNA were confirmed by long-range PCR (Expand Long Template PCR System, Roche Applied Science), conducted in accordance with the manufacturer's instructions.

    Techniques: Polymerase Chain Reaction, Sequencing, Negative Control, Marker

    LCT13 and TFPI-2as expression is linked. ( A ) Schematic diagram of the genomic region in Figure 1 A indicating regions (1–7) analysed by strand-specific RT–PCR (middle). Shown above and below the schematic are the ethidium bromide–stained gels used to visualize the strand-specific RT–PCR. Regions 2–7 are specifically expressed in cancer cell lines (H, HCC-1954 and M, MCF-7), but not normal breast (N), showing that cancer-specific antisense transcription is detectable up to 300 kb away from the TFPI-2 gene and up to the LINE-1 retrotransposon associated with LCT13. ( B ) siRNA knockdown of the LCT13 transcript. 2D densitometry of semiquantitative strand-specific RT–PCR analysis normalized to APRT control reveals an approximate 50% knockdown in LCT13 levels in cells transfected with a pool of three siRNA duplexes directed against LCT13 compared to those transfected with scrambled control siRNAs (left panel). This is paralleled by a 40–50% decrease in the TFPI-2as transcript (right panel).

    Journal: Nucleic Acids Research

    Article Title: Expression of a large LINE-1-driven antisense RNA is linked to epigenetic silencing of the metastasis suppressor gene TFPI-2 in cancer

    doi: 10.1093/nar/gkt438

    Figure Lengend Snippet: LCT13 and TFPI-2as expression is linked. ( A ) Schematic diagram of the genomic region in Figure 1 A indicating regions (1–7) analysed by strand-specific RT–PCR (middle). Shown above and below the schematic are the ethidium bromide–stained gels used to visualize the strand-specific RT–PCR. Regions 2–7 are specifically expressed in cancer cell lines (H, HCC-1954 and M, MCF-7), but not normal breast (N), showing that cancer-specific antisense transcription is detectable up to 300 kb away from the TFPI-2 gene and up to the LINE-1 retrotransposon associated with LCT13. ( B ) siRNA knockdown of the LCT13 transcript. 2D densitometry of semiquantitative strand-specific RT–PCR analysis normalized to APRT control reveals an approximate 50% knockdown in LCT13 levels in cells transfected with a pool of three siRNA duplexes directed against LCT13 compared to those transfected with scrambled control siRNAs (left panel). This is paralleled by a 40–50% decrease in the TFPI-2as transcript (right panel).

    Article Snippet: Generation of constructs and ES cell clones For pTFPI-2as and pTFPI-2pa constructs, a 4.93-kb human genomic DNA fragment including the full-length TFPI-2 gene obtained by long-range PCR (Expand Long PCR kit, Roche) on human genomic DNA with primers HC63f and HC63g and was cloned into the BamHI and KpnI sites of pcDNA3 (Invitrogen) and pcDNA3p(A)for, respectively. pcDNA3p(A)for was derived from pcDNA3 by cloning a 262 bp BGHp(A) fragment, obtained by PCR on pcDNA3 with primers Hind-p(A)-for and Hind-p(A)-rev, into the HindIII site of pcDNA3.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Transfection

    A human TFPI-2 transgene is sensitive to antisense RNA repression in mouse ES cells. (A) Schematic diagram of constructs introduced into mouse ES cells: pTFPI-2as is designed to transcribe antisense to TFPI-2 from a CMV promoter, while pTFPI-2pa has a poly-A signal insertion downstream of the CMV promoter to block antisense transcription. Arrows indicate direction of transcription. Regions analysed by ChIP are annotated as ‘prom’ and ‘ex-in2’. ( B ) Strand-specific RT–PCR analysis of TFPI-2 antisenese (TFPI-2as) expression in transgenic mouse ES cell lines demonstrates increased levels in pTFPI-2as lines (L2 and L12) relative to pTFPI-2pa cells (L7 and L9), mouse Aprt acts as a positive control for RNA quality and quantity. This correlates with a reduction in TFPI-2 expression as shown by real-time PCR normalized to mouse Gapdh . ( C ) ChIP analysis followed by real-time PCR. Left panel: Antibodies to H3K9me3 reveal localized enrichment of H3K9me3 in the promoter region in the antisense expressing cell line, pTFPI-2as (L2), compared to cells transfected with pTFPI-2pa (L9), which express low levels of TFPI-2as. Right panel: Antibodies to H4K20me3 also show enrichment at the TFPI-2 promoter in pTFPI-2as compared to pTFPI-2pa.

    Journal: Nucleic Acids Research

    Article Title: Expression of a large LINE-1-driven antisense RNA is linked to epigenetic silencing of the metastasis suppressor gene TFPI-2 in cancer

    doi: 10.1093/nar/gkt438

    Figure Lengend Snippet: A human TFPI-2 transgene is sensitive to antisense RNA repression in mouse ES cells. (A) Schematic diagram of constructs introduced into mouse ES cells: pTFPI-2as is designed to transcribe antisense to TFPI-2 from a CMV promoter, while pTFPI-2pa has a poly-A signal insertion downstream of the CMV promoter to block antisense transcription. Arrows indicate direction of transcription. Regions analysed by ChIP are annotated as ‘prom’ and ‘ex-in2’. ( B ) Strand-specific RT–PCR analysis of TFPI-2 antisenese (TFPI-2as) expression in transgenic mouse ES cell lines demonstrates increased levels in pTFPI-2as lines (L2 and L12) relative to pTFPI-2pa cells (L7 and L9), mouse Aprt acts as a positive control for RNA quality and quantity. This correlates with a reduction in TFPI-2 expression as shown by real-time PCR normalized to mouse Gapdh . ( C ) ChIP analysis followed by real-time PCR. Left panel: Antibodies to H3K9me3 reveal localized enrichment of H3K9me3 in the promoter region in the antisense expressing cell line, pTFPI-2as (L2), compared to cells transfected with pTFPI-2pa (L9), which express low levels of TFPI-2as. Right panel: Antibodies to H4K20me3 also show enrichment at the TFPI-2 promoter in pTFPI-2as compared to pTFPI-2pa.

    Article Snippet: Generation of constructs and ES cell clones For pTFPI-2as and pTFPI-2pa constructs, a 4.93-kb human genomic DNA fragment including the full-length TFPI-2 gene obtained by long-range PCR (Expand Long PCR kit, Roche) on human genomic DNA with primers HC63f and HC63g and was cloned into the BamHI and KpnI sites of pcDNA3 (Invitrogen) and pcDNA3p(A)for, respectively. pcDNA3p(A)for was derived from pcDNA3 by cloning a 262 bp BGHp(A) fragment, obtained by PCR on pcDNA3 with primers Hind-p(A)-for and Hind-p(A)-rev, into the HindIII site of pcDNA3.

    Techniques: Construct, Blocking Assay, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Expressing, Transgenic Assay, Positive Control, Real-time Polymerase Chain Reaction, Transfection

    Correlated expression of LCT13 and TFPI-2as transcripts in breast cancer cells. ( A ) Schematic diagram of a 300-kb region of chromosome 7q21.3 including LCT13 and the TFPI-2 gene. Scale is kilobase and indicates the position from the centromere with the value of 0 arbitrarily assigned to the TSS of CALCR . Genes (5′ segment of CALCR , TFPI-2 and GNGT1 ) are indicated as gray arrows. Two LINE-1 elements are present in the region (L1PA2 and L1PA6). Transcriptional orientations are indicated by arrows. LCT13 is a previously identified transcript shown to initiate at an L1ASP by 5′ RACE ( 22 ). TFPI-2as is the fragment analysed by strand-specific RT–PCR to test for the presence of TFPI-2 antisense RNAs. Displayed are the three spliced ESTs isolated from kidney (BG432114) and liver (DW466562 and DW435092) libraries that initiate at the LINE1 antisense promoter like LCT13 and extend past the TFPI-2 gene with a putative alternative transcript GNGT1-005 also annotated. ( B ) Expression of TFPI-2as (upper) and TFPI-2 (lower) in normal breast (N) and in breast cancer cell lines (H, HCC-1954; M, MCF7) analysed by strand specific and real-time RT–PCR, respectively. TFPI-2 expression is reduced in both breast cancer cell lines compared to normal controls (n = 3). TFPI-2 expression levels were normalized to HPRT . ( C ) Expression of TFPI-2as (upper) and TFPI-2 (lower) in a panel of five matched normal and tumour breast tissue analysed as described in B.

    Journal: Nucleic Acids Research

    Article Title: Expression of a large LINE-1-driven antisense RNA is linked to epigenetic silencing of the metastasis suppressor gene TFPI-2 in cancer

    doi: 10.1093/nar/gkt438

    Figure Lengend Snippet: Correlated expression of LCT13 and TFPI-2as transcripts in breast cancer cells. ( A ) Schematic diagram of a 300-kb region of chromosome 7q21.3 including LCT13 and the TFPI-2 gene. Scale is kilobase and indicates the position from the centromere with the value of 0 arbitrarily assigned to the TSS of CALCR . Genes (5′ segment of CALCR , TFPI-2 and GNGT1 ) are indicated as gray arrows. Two LINE-1 elements are present in the region (L1PA2 and L1PA6). Transcriptional orientations are indicated by arrows. LCT13 is a previously identified transcript shown to initiate at an L1ASP by 5′ RACE ( 22 ). TFPI-2as is the fragment analysed by strand-specific RT–PCR to test for the presence of TFPI-2 antisense RNAs. Displayed are the three spliced ESTs isolated from kidney (BG432114) and liver (DW466562 and DW435092) libraries that initiate at the LINE1 antisense promoter like LCT13 and extend past the TFPI-2 gene with a putative alternative transcript GNGT1-005 also annotated. ( B ) Expression of TFPI-2as (upper) and TFPI-2 (lower) in normal breast (N) and in breast cancer cell lines (H, HCC-1954; M, MCF7) analysed by strand specific and real-time RT–PCR, respectively. TFPI-2 expression is reduced in both breast cancer cell lines compared to normal controls (n = 3). TFPI-2 expression levels were normalized to HPRT . ( C ) Expression of TFPI-2as (upper) and TFPI-2 (lower) in a panel of five matched normal and tumour breast tissue analysed as described in B.

    Article Snippet: Generation of constructs and ES cell clones For pTFPI-2as and pTFPI-2pa constructs, a 4.93-kb human genomic DNA fragment including the full-length TFPI-2 gene obtained by long-range PCR (Expand Long PCR kit, Roche) on human genomic DNA with primers HC63f and HC63g and was cloned into the BamHI and KpnI sites of pcDNA3 (Invitrogen) and pcDNA3p(A)for, respectively. pcDNA3p(A)for was derived from pcDNA3 by cloning a 262 bp BGHp(A) fragment, obtained by PCR on pcDNA3 with primers Hind-p(A)-for and Hind-p(A)-rev, into the HindIII site of pcDNA3.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Quantitative RT-PCR

    PCR amplification of fnr region from different MG1655 isolates. The fnr region was amplified from the CGSC isolate of MG1655 (CGSC 6300; lane 1) and the isolate obtained from M. Singer and C. Gross (NCM3430; lane 2) (see Materials and Methods). The sizes of the molecular standards in lane 3 are noted to the right. The genes deleted in the CGSC isolate (b1332 to b1344) are, respectively, ynaJ (open reading frame conserved in E. coli and Salmonella enterica ), uspE ( ydaA ), fnr (Crp family activator of anaerobic respiratory gene transcription), ogt ( O -6-alkylguanine-DNA/cysteine-protein methyltransferase), abgT ( ydaH ; p ), abgB ( ydaI ; p ), abgA ( ydaJ ; p ), abgR ( ydaK ; p ), ydaL (open reading frame conserved in enterobacteria), ydaM (open reading frame conserved in E. coli ), ydaN (open reading frame conserved in enterobacteria), dbpA (ATP-dependent RNA helicase), and ydaO (open reading frame conserved in enterobacteria). The deletion is flanked by tns5_4 (b1331), which codes for IS 5 transposase, and ydaP (b1345), a rac prophage which codes for a putative prophage integrase.

    Journal: Journal of Bacteriology

    Article Title: Physiological Studies of Escherichia coli Strain MG1655: Growth Defects and Apparent Cross-Regulation of Gene Expression

    doi: 10.1128/JB.185.18.5611-5626.2003

    Figure Lengend Snippet: PCR amplification of fnr region from different MG1655 isolates. The fnr region was amplified from the CGSC isolate of MG1655 (CGSC 6300; lane 1) and the isolate obtained from M. Singer and C. Gross (NCM3430; lane 2) (see Materials and Methods). The sizes of the molecular standards in lane 3 are noted to the right. The genes deleted in the CGSC isolate (b1332 to b1344) are, respectively, ynaJ (open reading frame conserved in E. coli and Salmonella enterica ), uspE ( ydaA ), fnr (Crp family activator of anaerobic respiratory gene transcription), ogt ( O -6-alkylguanine-DNA/cysteine-protein methyltransferase), abgT ( ydaH ; p ), abgB ( ydaI ; p ), abgA ( ydaJ ; p ), abgR ( ydaK ; p ), ydaL (open reading frame conserved in enterobacteria), ydaM (open reading frame conserved in E. coli ), ydaN (open reading frame conserved in enterobacteria), dbpA (ATP-dependent RNA helicase), and ydaO (open reading frame conserved in enterobacteria). The deletion is flanked by tns5_4 (b1331), which codes for IS 5 transposase, and ydaP (b1345), a rac prophage which codes for a putative prophage integrase.

    Article Snippet: The entire fnr region was amplified by long-range PCR according to the manufacturer's instructions (Expand PCR Long Range; Boehringer-Roche) with 40 ng of genomic DNA, 3.75 U of Expand polymerase, 1× reaction buffer 2, 500 μM deoxynucleoside triphosphate mix, and 300 nM each primer in a 50-μl reaction) with forward primer b1331 and reverse primer b1345 at 94°C for 120 s and then for 10 cycles at 94°C for 10 s, 60°C for 30 s, and 68°C for 15 min, and 15 additional cycles with an increase of 20 s per cycle at 68°C (see Fig. ).

    Techniques: Polymerase Chain Reaction, Amplification

    Deletion of SAMHD1 does not affect mtDNA stability. a) MtDNA copy number in the TA muscle of 5 or 6 wt (filled dots) and SAMHD1 −/− (open dots) 13-week-old (adult), 1-year-old (old adult), and 2-year-old (aged) animals was determined by qPCR and normalized to the value for adult wt mice. The mean for each group is indicated by a horizontal line. The p-values were calculated using Welch’s t-test; ns, non-significant. b) DNA isolated from embryos and from the TA muscle of pups, adults, 1-year-old (old) adults, or aged animals was linearized with SacI endonuclease and separated on a neutral gel. MtDNA was visualized as above. Full-length mtDNA is indicated (FL); the asterisk denotes a higher-migrating species resistant to cleavage. c) Long-range PCR to detect deletions in mtDNA from the TA muscle of wt mice of various ages. Full-length product is indicated (FL). Only minor species containing deletions are observed in the mtDNA from old adults and aged animals, as indicated by the vertical line on the right-hand side of the gel. d) Untreated or alkali-treated DNA from skeletal muscle of aged wt and SAMHD1 −/− (ko) mice was analyzed on a denaturing gel, and mtDNA was visualized using a COX1 probe. Each sample lane corresponds to an individual mouse, and dotted lines represent the median. e) The median length of the untreated mtDNA in samples from Fig. 5d is indicated by a horizontal line. The two groups were compared using Welch’s t-test (ns, non-significant; n = 4). f) The length difference between untreated and alkali-treated mtDNAs shown in Fig. 5d was used to compute the number of rNMPs per single strand of mtDNA. The horizontal lines indicate the median. The p-value of the statistically significant difference between the two groups was calculated by Welch’s t-test; n = 4. g) Long-range PCR was performed on mtDNA isolated from the TA muscle of adult and aged wt or SAMHD1 −/− (ko) mice. FL, full-length product; the vertical line indicates the size range of mtDNA molecules with deletions. h) Kaplan–Meier survival curve for wt and SAMHD1 −/− (ko) mice. Comparison of the curves by the log-rank (Mantel–Cox) test confirmed no statistically significant difference between the genotypes. The sizes of the bands in the DNA ladder are indicated in kb. See also Fig. S5.

    Journal: bioRxiv

    Article Title: The physiological level of rNMPs present in mtDNA does not compromise its stability

    doi: 10.1101/746719

    Figure Lengend Snippet: Deletion of SAMHD1 does not affect mtDNA stability. a) MtDNA copy number in the TA muscle of 5 or 6 wt (filled dots) and SAMHD1 −/− (open dots) 13-week-old (adult), 1-year-old (old adult), and 2-year-old (aged) animals was determined by qPCR and normalized to the value for adult wt mice. The mean for each group is indicated by a horizontal line. The p-values were calculated using Welch’s t-test; ns, non-significant. b) DNA isolated from embryos and from the TA muscle of pups, adults, 1-year-old (old) adults, or aged animals was linearized with SacI endonuclease and separated on a neutral gel. MtDNA was visualized as above. Full-length mtDNA is indicated (FL); the asterisk denotes a higher-migrating species resistant to cleavage. c) Long-range PCR to detect deletions in mtDNA from the TA muscle of wt mice of various ages. Full-length product is indicated (FL). Only minor species containing deletions are observed in the mtDNA from old adults and aged animals, as indicated by the vertical line on the right-hand side of the gel. d) Untreated or alkali-treated DNA from skeletal muscle of aged wt and SAMHD1 −/− (ko) mice was analyzed on a denaturing gel, and mtDNA was visualized using a COX1 probe. Each sample lane corresponds to an individual mouse, and dotted lines represent the median. e) The median length of the untreated mtDNA in samples from Fig. 5d is indicated by a horizontal line. The two groups were compared using Welch’s t-test (ns, non-significant; n = 4). f) The length difference between untreated and alkali-treated mtDNAs shown in Fig. 5d was used to compute the number of rNMPs per single strand of mtDNA. The horizontal lines indicate the median. The p-value of the statistically significant difference between the two groups was calculated by Welch’s t-test; n = 4. g) Long-range PCR was performed on mtDNA isolated from the TA muscle of adult and aged wt or SAMHD1 −/− (ko) mice. FL, full-length product; the vertical line indicates the size range of mtDNA molecules with deletions. h) Kaplan–Meier survival curve for wt and SAMHD1 −/− (ko) mice. Comparison of the curves by the log-rank (Mantel–Cox) test confirmed no statistically significant difference between the genotypes. The sizes of the bands in the DNA ladder are indicated in kb. See also Fig. S5.

    Article Snippet: MtDNA deletion analysis by long-range PCR The Expand Long Template PCR system (Roche) with forward and reverse primers at nt 2,478–2,512 and nt 1,933–1,906, respectively, was used to amplify a ~15,800 bp fragment of mouse mtDNA from 25 ng of total DNA.

    Techniques: Real-time Polymerase Chain Reaction, Mouse Assay, Isolation, Polymerase Chain Reaction