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 2162 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 2162 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 "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

    4) 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

    5) 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

    6) 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

    7) 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

    8) 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

    9) 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

    10) 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

    11) 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

    12) 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

    13) 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

    14) 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:

    15) 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

    16) 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

    17) 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

    18) 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

    19) 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

    20) Product Images from "An RNA-targeted therapy for dystrophic epidermolysis bullosa"

    Article Title: An RNA-targeted therapy for dystrophic epidermolysis bullosa

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx669

    Analysis of the trans -spliced COL7A1 mRNA in the LV-RTM-S6m corrected RDEB single cell clone C47. ( A ) Via sqRT-PCR using primers specifically hybridizing to endogenous COL7A1 exon 62/63 and the introduced silent mutation in exon 65 on the RTM, we detected a product of 216 bp, corresponding to the trans -spliced COL7A1 mRNA in LV-RTM-S6m transduced RDEB cell pool and single cell clone C47. LV-RTM-woBD transduced cells served as negative control. ( B ) Sequence analysis of trans-spliced COL7A1 mRNA shows the correct exon 64/65 junction as well as the amplified silent mutations. ( C ) SqRT-PCR analysis showed that 2.113% of COL7A1 mRNA in C47 was correctly trans -spliced. Mean of four individual experiments and error bars (SEM). ***P-value
    Figure Legend Snippet: Analysis of the trans -spliced COL7A1 mRNA in the LV-RTM-S6m corrected RDEB single cell clone C47. ( A ) Via sqRT-PCR using primers specifically hybridizing to endogenous COL7A1 exon 62/63 and the introduced silent mutation in exon 65 on the RTM, we detected a product of 216 bp, corresponding to the trans -spliced COL7A1 mRNA in LV-RTM-S6m transduced RDEB cell pool and single cell clone C47. LV-RTM-woBD transduced cells served as negative control. ( B ) Sequence analysis of trans-spliced COL7A1 mRNA shows the correct exon 64/65 junction as well as the amplified silent mutations. ( C ) SqRT-PCR analysis showed that 2.113% of COL7A1 mRNA in C47 was correctly trans -spliced. Mean of four individual experiments and error bars (SEM). ***P-value

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

    21) 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

    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

    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

    22) Product Images from "A distal enhancer controls cytokine-dependent human cPLA2? gene expression [S]"

    Article Title: A distal enhancer controls cytokine-dependent human cPLA2? gene expression [S]

    Journal: Journal of Lipid Research

    doi: 10.1194/jlr.M037382

    IL-1β induction and chromatin structure of cPLA 2 α in HFL-1. (A, left) HFL-1 cells were treated with 2 ng/µl IL-1β for the indicated times, and total RNA was analyzed by real-time RT-PCR. Relative fold induction was calculated
    Figure Legend Snippet: IL-1β induction and chromatin structure of cPLA 2 α in HFL-1. (A, left) HFL-1 cells were treated with 2 ng/µl IL-1β for the indicated times, and total RNA was analyzed by real-time RT-PCR. Relative fold induction was calculated

    Techniques Used: Quantitative RT-PCR

    23) Product Images from "Horizontal Gene Transfer and Redundancy of Tryptophan Biosynthetic Enzymes in Dinotoms"

    Article Title: Horizontal Gene Transfer and Redundancy of Tryptophan Biosynthetic Enzymes in Dinotoms

    Journal: Genome Biology and Evolution

    doi: 10.1093/gbe/evu014

    The distribution of the GC content of the diatom and dinotom sequences. ( A ) The distribution of the GC content of all the EST sequences > 150 bp available for three diatoms, downloaded from the National Center for Biotechnology Information EST database on December 4, 2013. Fc, Fragilariopsis cylindrus ; Pt, Phaeodactylum tricornutum ; Tp, Thalassiosira pseudonana . ( B ) The distribution of the GC content of the dinotom total mRNA and SL cDNA sequences. The x axis shows the GC content, and the y axis the number of sequences. DbD, Durinskia baltica dark sample; DbL, D. baltica light sample; GfD, Glenodinium foliaceum dark sample; GfL, G. foliaceum light sample; KfD, Kryptoperidinium foliaceum dark sample; KfL, K. foliaceum light sample; DbSLcDNA, D. baltica SL cDNA library.
    Figure Legend Snippet: The distribution of the GC content of the diatom and dinotom sequences. ( A ) The distribution of the GC content of all the EST sequences > 150 bp available for three diatoms, downloaded from the National Center for Biotechnology Information EST database on December 4, 2013. Fc, Fragilariopsis cylindrus ; Pt, Phaeodactylum tricornutum ; Tp, Thalassiosira pseudonana . ( B ) The distribution of the GC content of the dinotom total mRNA and SL cDNA sequences. The x axis shows the GC content, and the y axis the number of sequences. DbD, Durinskia baltica dark sample; DbL, D. baltica light sample; GfD, Glenodinium foliaceum dark sample; GfL, G. foliaceum light sample; KfD, Kryptoperidinium foliaceum dark sample; KfL, K. foliaceum light sample; DbSLcDNA, D. baltica SL cDNA library.

    Techniques Used: cDNA Library Assay

    The maximum likelihood trees for the enzymes of the tryptophan biosynthetic pathway in dinoflagellates. ( A ) Anthranilate synthase (AS) phylogeny, partial tree. ( B ) Anthranilate phosphoribosyltransferase (PRT). ( C ) Indole-3-glycerol-phosphate synthase and phosphoribosylanthranilate isomerase fusion (InGPS-PRAI) phylogeny. ( D ) Tryptophan synthase (TS) phylogeny, partial tree. Numbers at the nodes indicate the bootstrap support ≥ 50 for the majority of the nodes. The dinotom clades are highlighted with boxes in green (with diatoms) and cream (with dinoflagellates). The dinotom sequences with a low or high GC content are shown in red or turquoise fonts, respectively. Some major groups are also color coded: diatoms in purple font; other stramenopiles in brown; streptophytes and green algae in green; red algae in scarlet; dinoflagellates in blue; and fungi in orange. All other groups are in black font, and with the exception of prokaryotes, the name of the group appears before the species name. The accession numbers are given in the supplementary file S1 , Supplementary Material online. Db-D/L, Durinskia baltica dark/light; Kf-D/L, Kryptoperidinium foliaceum dark/light; Gf-D/L, Glenodinium foliaceum dark/light.
    Figure Legend Snippet: The maximum likelihood trees for the enzymes of the tryptophan biosynthetic pathway in dinoflagellates. ( A ) Anthranilate synthase (AS) phylogeny, partial tree. ( B ) Anthranilate phosphoribosyltransferase (PRT). ( C ) Indole-3-glycerol-phosphate synthase and phosphoribosylanthranilate isomerase fusion (InGPS-PRAI) phylogeny. ( D ) Tryptophan synthase (TS) phylogeny, partial tree. Numbers at the nodes indicate the bootstrap support ≥ 50 for the majority of the nodes. The dinotom clades are highlighted with boxes in green (with diatoms) and cream (with dinoflagellates). The dinotom sequences with a low or high GC content are shown in red or turquoise fonts, respectively. Some major groups are also color coded: diatoms in purple font; other stramenopiles in brown; streptophytes and green algae in green; red algae in scarlet; dinoflagellates in blue; and fungi in orange. All other groups are in black font, and with the exception of prokaryotes, the name of the group appears before the species name. The accession numbers are given in the supplementary file S1 , Supplementary Material online. Db-D/L, Durinskia baltica dark/light; Kf-D/L, Kryptoperidinium foliaceum dark/light; Gf-D/L, Glenodinium foliaceum dark/light.

    Techniques Used:

    The maximum likelihood trees for the enzymes of the tryptophan biosynthetic pathway in dinotoms. ( A ) Anthranilate synthase (AS) phylogeny, partial tree. ( B ) Anthranilate phosphoribosyltransferase (PRT), partial tree. ( C ) Indole-3-glycerol-phosphate synthase and phosphoribosylanthranilate isomerase fusion (InGPS-PRAI) phylogeny. ( D ) Tryptophan synthase (TS) phylogeny, partial tree. Numbers at the nodes indicate the bootstrap support ≥ 50 for the majority of the nodes. The dinotom clades are highlighted with boxes in green (with diatoms) and cream. The numbers next to dinotom taxa indicate the GC content of their protein-coding transcripts. The checkmarks indicate the fusion proteins in dinotoms. The black dots denote the presence of an SP as predicted by SignalP 3.0 ( Bendtsen et al. 2004 ). The dinotom sequences with a low or high GC content are shown in red or turquoise fonts, respectively. Some major groups are also color coded: diatoms in purple font; other stramenopiles in brown; streptophytes and green algae in green; red algae in scarlet; dinoflagellates in blue; and fungi in orange. All other groups are in black font, and with the exception of prokaryotes, the name of the group appears before the species name. The accession numbers are given in the supplementary file S1 , Supplementary Material online. Db-D/L, Durinskia baltica dark/light; Kf-D/L, Kryptoperidinium foliaceum dark/light; Gf-D/L, Glenodinium foliaceum dark/light.
    Figure Legend Snippet: The maximum likelihood trees for the enzymes of the tryptophan biosynthetic pathway in dinotoms. ( A ) Anthranilate synthase (AS) phylogeny, partial tree. ( B ) Anthranilate phosphoribosyltransferase (PRT), partial tree. ( C ) Indole-3-glycerol-phosphate synthase and phosphoribosylanthranilate isomerase fusion (InGPS-PRAI) phylogeny. ( D ) Tryptophan synthase (TS) phylogeny, partial tree. Numbers at the nodes indicate the bootstrap support ≥ 50 for the majority of the nodes. The dinotom clades are highlighted with boxes in green (with diatoms) and cream. The numbers next to dinotom taxa indicate the GC content of their protein-coding transcripts. The checkmarks indicate the fusion proteins in dinotoms. The black dots denote the presence of an SP as predicted by SignalP 3.0 ( Bendtsen et al. 2004 ). The dinotom sequences with a low or high GC content are shown in red or turquoise fonts, respectively. Some major groups are also color coded: diatoms in purple font; other stramenopiles in brown; streptophytes and green algae in green; red algae in scarlet; dinoflagellates in blue; and fungi in orange. All other groups are in black font, and with the exception of prokaryotes, the name of the group appears before the species name. The accession numbers are given in the supplementary file S1 , Supplementary Material online. Db-D/L, Durinskia baltica dark/light; Kf-D/L, Kryptoperidinium foliaceum dark/light; Gf-D/L, Glenodinium foliaceum dark/light.

    Techniques Used:

    24) Product Images from "An RNA-targeted therapy for dystrophic epidermolysis bullosa"

    Article Title: An RNA-targeted therapy for dystrophic epidermolysis bullosa

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx669

    LV-RTM-S6m transduced RDEB patient keratinocytes showed trans -splicing corrected type VII collagen expression. ( A ) The bidirectional SIN lentiviral vector contained the coding sequence of COL7A1 exons 65–118 and the 224 bp BD in antisense direction expressed by a spleen focus forming virus promoter (SF-Pro). The GFP and puromycin cassette (Puro) under the control of a mnCMV promoter was inserted in sense direction. ( B ) Stable transduction of LV-RTM-S6m into an RDEB keratinocyte cell line induced accurate trans -splicing into the endogenous COL7A1 pre-mRNA, leading to the replacement of endogenous COL7A1 exons 65–118 by a wild-type copy provided by the RTM. Asterisk = silent mutations. ( C ) PCR analysis on genomic DNA of LV-RTM-S6m and LV-RTMm-woBD transduced RDEB keratinocytes showed full-length genomic RTM integration (3.6 kb). Positive control: LV-RTM-S6m plasmid. ( D ) Immunofluoresence staining showed a specific signal indicating type VII collagen expression (red) in LV-RTM-S6m transduced RDEB keratinocytes, similar to healthy wild-type keratinocytes (normal kc) and hardly visible in negative controls (untransduced and LV-RTMm-woBD). Cell nuclei: 4′,6-diamidin-2-phenylindol (DAPI, blue). Scale bar (SB) = 20 μm.
    Figure Legend Snippet: LV-RTM-S6m transduced RDEB patient keratinocytes showed trans -splicing corrected type VII collagen expression. ( A ) The bidirectional SIN lentiviral vector contained the coding sequence of COL7A1 exons 65–118 and the 224 bp BD in antisense direction expressed by a spleen focus forming virus promoter (SF-Pro). The GFP and puromycin cassette (Puro) under the control of a mnCMV promoter was inserted in sense direction. ( B ) Stable transduction of LV-RTM-S6m into an RDEB keratinocyte cell line induced accurate trans -splicing into the endogenous COL7A1 pre-mRNA, leading to the replacement of endogenous COL7A1 exons 65–118 by a wild-type copy provided by the RTM. Asterisk = silent mutations. ( C ) PCR analysis on genomic DNA of LV-RTM-S6m and LV-RTMm-woBD transduced RDEB keratinocytes showed full-length genomic RTM integration (3.6 kb). Positive control: LV-RTM-S6m plasmid. ( D ) Immunofluoresence staining showed a specific signal indicating type VII collagen expression (red) in LV-RTM-S6m transduced RDEB keratinocytes, similar to healthy wild-type keratinocytes (normal kc) and hardly visible in negative controls (untransduced and LV-RTMm-woBD). Cell nuclei: 4′,6-diamidin-2-phenylindol (DAPI, blue). Scale bar (SB) = 20 μm.

    Techniques Used: Expressing, Plasmid Preparation, Sequencing, Transduction, Polymerase Chain Reaction, Positive Control, Staining

    Analysis of the trans -spliced COL7A1 mRNA in the LV-RTM-S6m corrected RDEB single cell clone C47. ( A ) Via sqRT-PCR using primers specifically hybridizing to endogenous COL7A1 exon 62/63 and the introduced silent mutation in exon 65 on the RTM, we detected a product of 216 bp, corresponding to the trans -spliced COL7A1 mRNA in LV-RTM-S6m transduced RDEB cell pool and single cell clone C47. LV-RTM-woBD transduced cells served as negative control. ( B ) Sequence analysis of trans-spliced COL7A1 mRNA shows the correct exon 64/65 junction as well as the amplified silent mutations. ( C ) SqRT-PCR analysis showed that 2.113% of COL7A1 mRNA in C47 was correctly trans -spliced. Mean of four individual experiments and error bars (SEM). ***P-value
    Figure Legend Snippet: Analysis of the trans -spliced COL7A1 mRNA in the LV-RTM-S6m corrected RDEB single cell clone C47. ( A ) Via sqRT-PCR using primers specifically hybridizing to endogenous COL7A1 exon 62/63 and the introduced silent mutation in exon 65 on the RTM, we detected a product of 216 bp, corresponding to the trans -spliced COL7A1 mRNA in LV-RTM-S6m transduced RDEB cell pool and single cell clone C47. LV-RTM-woBD transduced cells served as negative control. ( B ) Sequence analysis of trans-spliced COL7A1 mRNA shows the correct exon 64/65 junction as well as the amplified silent mutations. ( C ) SqRT-PCR analysis showed that 2.113% of COL7A1 mRNA in C47 was correctly trans -spliced. Mean of four individual experiments and error bars (SEM). ***P-value

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

    25) Product Images from "Next-Generation Sequencing Identifies the Danforth's Short Tail Mouse Mutation as a Retrotransposon Insertion Affecting Ptf1a ExpressionEctopic Expression of Ptf1a Induces Spinal Defects, Urogenital Defects and Anorectal Malformations in Danforth's Short Tail MiceA Retrotransposon Insertion in the 5′ Regulatory Domain of Ptf1a Results in Ectopic Gene Expression and Multiple Congenital Defects in Danforth's Short Tail MouseRetrotransposon Activates Ectopic Ptf1a Expression: A Short Tail"

    Article Title: Next-Generation Sequencing Identifies the Danforth's Short Tail Mouse Mutation as a Retrotransposon Insertion Affecting Ptf1a ExpressionEctopic Expression of Ptf1a Induces Spinal Defects, Urogenital Defects and Anorectal Malformations in Danforth's Short Tail MiceA Retrotransposon Insertion in the 5′ Regulatory Domain of Ptf1a Results in Ectopic Gene Expression and Multiple Congenital Defects in Danforth's Short Tail MouseRetrotransposon Activates Ectopic Ptf1a Expression: A Short Tail

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003205

    Confirmation and mapping of the Sd mutation. A) Southern analysis and PCR showing the presence of a large DNA insertion at the Sd locus. B) Multiplex PCR from inbred mouse lines showing the mutation is only present in Sd mice, and is not a polymorphism. In this three primer PCR reaction the Sd amplimer is 406 bp, while the WT amplimer is 510 bp. C) Mapping of the Sd mutation which we identified as an ETn (early transposon) in relation to nearby gene/ESTs, figure not to scale.
    Figure Legend Snippet: Confirmation and mapping of the Sd mutation. A) Southern analysis and PCR showing the presence of a large DNA insertion at the Sd locus. B) Multiplex PCR from inbred mouse lines showing the mutation is only present in Sd mice, and is not a polymorphism. In this three primer PCR reaction the Sd amplimer is 406 bp, while the WT amplimer is 510 bp. C) Mapping of the Sd mutation which we identified as an ETn (early transposon) in relation to nearby gene/ESTs, figure not to scale.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Multiplex Assay, Mouse Assay

    26) 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

    27) 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

    Effect of drug treatment on BKPy viral gene expression in human salivary gland and Vero cells HSG ( A and B ) and Vero ( C and D ) cells were infected with BKPyV for 24h then treated with drug as described in the materials and methods. At stated times post infection cells were collected, RNA isolated, cDNA generated and qRTPCR performed for T Ag ( A and C ) or VP1 ( B and D ) viral transcripts. Gene expression values were normalized to the levels of β-actin transcripts, using 2 −DDC(T) method and are represented as the changes (n-fold) in transcript levels with the levels in non-drug treated (BKPYV only) samples arbitrarily set to 1. At stated times post infection cell lysates were collected and used for immunoblotting as described in the materials and methods. Antibodies against T Ag and β-actin ( E ) were used ( F ). Relative L Tag protein expression compared to β-actin.
    Figure Legend Snippet: Effect of drug treatment on BKPy viral gene expression in human salivary gland and Vero cells HSG ( A and B ) and Vero ( C and D ) cells were infected with BKPyV for 24h then treated with drug as described in the materials and methods. At stated times post infection cells were collected, RNA isolated, cDNA generated and qRTPCR performed for T Ag ( A and C ) or VP1 ( B and D ) viral transcripts. Gene expression values were normalized to the levels of β-actin transcripts, using 2 −DDC(T) method and are represented as the changes (n-fold) in transcript levels with the levels in non-drug treated (BKPYV only) samples arbitrarily set to 1. At stated times post infection cell lysates were collected and used for immunoblotting as described in the materials and methods. Antibodies against T Ag and β-actin ( E ) were used ( F ). Relative L Tag protein expression compared to β-actin.

    Techniques Used: Expressing, Infection, Isolation, Generated

    28) Product Images from "Transgene Silencing and Transgene-Derived siRNA Production in Tobacco Plants Homozygous for an Introduced AtMYB90 Construct"

    Article Title: Transgene Silencing and Transgene-Derived siRNA Production in Tobacco Plants Homozygous for an Introduced AtMYB90 Construct

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030141

    Graphic map of TDNA containing the 35S:: AtMYB90 construct. The main map shown represents the TDNA construct (containing a 35S:: AtMYB90 transgene) used to transform N. tabacum . Flagged vertical lines indicate TDNA/plant DNA junctions cloned from the Myb27 line, with plant segments shown above the main map ( NtL , tobacco sequence at the TR border; NtR-B1 and NtR-B2 , tobacco sequences at the TL border). Segments of the original plasmid TDNA that are absent at the Myb27 locus are shown as a faded graphic. Labels include: TR (right TDNA border); PClSV-Pro (Peanut Chlorotic Streak Virus promoter); BAR (basta resistance coding region); 35S-Ter (CaMV 35S transcription termination signal); 35S-Pro (CaMV 35S promoter with duplicated enhancer region [ Enh-1 , Enh-2 ]); AtMYB90 ( AtMYB90 coding region [the Myb R2–R3 repeats are indicated]); g7-Ter (transcription termination signal from the gene-7 of octopine TDNA); TL (left TDNA border). The locations of primers used for qRTPCR are indicated by blue arrowheads and the segment of the AtMYB90 coding region used in a hairpin expression vector (pKOihpMyb) is indicated by a red box. Dashed lines show the boundaries of PCR amplimers used to confirm the structure of the T-DNA insert (“ + ” indicates predicted PCR product observed, “ − ” indicates no product of the predicted size; primer sequences are listed in Additional files, Table S1 ). The PCR templates used were total plant leaf DNA from the Myb27 line and from untransformed tobacco, Nt (SR1) . The BAR and Myb probes used for blot hybridization are indicated by green boxes above the map and restriction enzyme cleavage sites used for Southern blot analysis, including resulting fragment sizes, are shown below the main map.
    Figure Legend Snippet: Graphic map of TDNA containing the 35S:: AtMYB90 construct. The main map shown represents the TDNA construct (containing a 35S:: AtMYB90 transgene) used to transform N. tabacum . Flagged vertical lines indicate TDNA/plant DNA junctions cloned from the Myb27 line, with plant segments shown above the main map ( NtL , tobacco sequence at the TR border; NtR-B1 and NtR-B2 , tobacco sequences at the TL border). Segments of the original plasmid TDNA that are absent at the Myb27 locus are shown as a faded graphic. Labels include: TR (right TDNA border); PClSV-Pro (Peanut Chlorotic Streak Virus promoter); BAR (basta resistance coding region); 35S-Ter (CaMV 35S transcription termination signal); 35S-Pro (CaMV 35S promoter with duplicated enhancer region [ Enh-1 , Enh-2 ]); AtMYB90 ( AtMYB90 coding region [the Myb R2–R3 repeats are indicated]); g7-Ter (transcription termination signal from the gene-7 of octopine TDNA); TL (left TDNA border). The locations of primers used for qRTPCR are indicated by blue arrowheads and the segment of the AtMYB90 coding region used in a hairpin expression vector (pKOihpMyb) is indicated by a red box. Dashed lines show the boundaries of PCR amplimers used to confirm the structure of the T-DNA insert (“ + ” indicates predicted PCR product observed, “ − ” indicates no product of the predicted size; primer sequences are listed in Additional files, Table S1 ). The PCR templates used were total plant leaf DNA from the Myb27 line and from untransformed tobacco, Nt (SR1) . The BAR and Myb probes used for blot hybridization are indicated by green boxes above the map and restriction enzyme cleavage sites used for Southern blot analysis, including resulting fragment sizes, are shown below the main map.

    Techniques Used: Construct, Clone Assay, Sequencing, Plasmid Preparation, Expressing, Polymerase Chain Reaction, Hybridization, Southern Blot

    29) 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

    30) Product Images from "Expanding the Host Range of Hepatitis C Virus through Viral Adaptation"

    Article Title: Expanding the Host Range of Hepatitis C Virus through Viral Adaptation

    Journal: mBio

    doi: 10.1128/mBio.01915-16

    Infection of mice with blunted innate immunity does not result in persistent infection. Mice with a fully intact innate immune system (wild-type [WT]) or lacking STAT1 were infected with 5 × 10 6 TCID 50 of Jc1 or Jc1/mCD81. HCV RNA levels (number of copies [cop] per milliliter) were measured by qRT-PCR at the indicated time points. Each symbol represents the value f or an individual animal. The dashed line shows the limit of detection (l.o.d.).
    Figure Legend Snippet: Infection of mice with blunted innate immunity does not result in persistent infection. Mice with a fully intact innate immune system (wild-type [WT]) or lacking STAT1 were infected with 5 × 10 6 TCID 50 of Jc1 or Jc1/mCD81. HCV RNA levels (number of copies [cop] per milliliter) were measured by qRT-PCR at the indicated time points. Each symbol represents the value f or an individual animal. The dashed line shows the limit of detection (l.o.d.).

    Techniques Used: Infection, Mouse Assay, Quantitative RT-PCR

    Uptake of mtHCV in vivo can be blocked with anti-HCV E2-specific antibodies and is dependent on endogenous expression of mouse CD81 and SCARB1. (a) R26-LSL-Fluc mice were injected with 1 × 10 11 particles of adenoviruses expressing mouse CD81, SCARB1, CLDN1, and OCLN. mtHCV-Cre was incubated with the indicated doses of anti-HCV E2 (clone AR4A) or an anti-HIV isotype control antibody (clone B12) for 1 h prior to injection (1 × 10 6 TCID 50 ) and 24 h after adenoviral delivery ( n = 3). Data were acquired 72 h following HCV infection. Values are means ± SD. (b) Rosa26-Fluc mice were crossed with mCD81 −/− or mSCARB1 −/− mice, and offspring with the indicated zygosities for the respective mutant alleles were injected with the indicated combinations of AdVs encoding mouse CD81 (mCD81), SCARB1, CLDN1, and OCLN and 24 h later with 2 × 10 7 TCID 50 BiCre-Jc1 ( n ≥ 4). Luminescence was quantified 72 h following HCV infection. Values are means plus SD.
    Figure Legend Snippet: Uptake of mtHCV in vivo can be blocked with anti-HCV E2-specific antibodies and is dependent on endogenous expression of mouse CD81 and SCARB1. (a) R26-LSL-Fluc mice were injected with 1 × 10 11 particles of adenoviruses expressing mouse CD81, SCARB1, CLDN1, and OCLN. mtHCV-Cre was incubated with the indicated doses of anti-HCV E2 (clone AR4A) or an anti-HIV isotype control antibody (clone B12) for 1 h prior to injection (1 × 10 6 TCID 50 ) and 24 h after adenoviral delivery ( n = 3). Data were acquired 72 h following HCV infection. Values are means ± SD. (b) Rosa26-Fluc mice were crossed with mCD81 −/− or mSCARB1 −/− mice, and offspring with the indicated zygosities for the respective mutant alleles were injected with the indicated combinations of AdVs encoding mouse CD81 (mCD81), SCARB1, CLDN1, and OCLN and 24 h later with 2 × 10 7 TCID 50 BiCre-Jc1 ( n ≥ 4). Luminescence was quantified 72 h following HCV infection. Values are means plus SD.

    Techniques Used: In Vivo, Expressing, Mouse Assay, Injection, Incubation, Infection, Mutagenesis

    31) Product Images from "An Expressed Retrogene of the Master Embryonic Stem Cell Gene POU5F1 Is Associated with Prostate Cancer Susceptibility"

    Article Title: An Expressed Retrogene of the Master Embryonic Stem Cell Gene POU5F1 Is Associated with Prostate Cancer Susceptibility

    Journal: American Journal of Human Genetics

    doi: 10.1016/j.ajhg.2014.01.019

    Two SNPs Are Predicted to Be Deleterious in POU5F1B
    Figure Legend Snippet: Two SNPs Are Predicted to Be Deleterious in POU5F1B

    Techniques Used:

    32) Product Images from "HIV infection reveals widespread expansion of novel centromeric human endogenous retroviruses"

    Article Title: HIV infection reveals widespread expansion of novel centromeric human endogenous retroviruses

    Journal: Genome Research

    doi: 10.1101/gr.144303.112

    Expansion of K111 proviruses in humans took place after the Homo-Pan divergence. ( A ) Detection of K111 full-length and solo LTR insertions from DNA of New and Old World primates. Full-length K111 proviruses were detected by PCR only in the human and chimpanzee.
    Figure Legend Snippet: Expansion of K111 proviruses in humans took place after the Homo-Pan divergence. ( A ) Detection of K111 full-length and solo LTR insertions from DNA of New and Old World primates. Full-length K111 proviruses were detected by PCR only in the human and chimpanzee.

    Techniques Used: Polymerase Chain Reaction

    HIV-1 infection, and HIV-1 Tat protein, activate K111 provirus expression in part by inducing loss of heterochromatin. HIV-1 infection of cell lines ( A ) or human peripheral blood lymphocytes (PBLs; B ) activates the expression of K111 as detected by qRT-PCR
    Figure Legend Snippet: HIV-1 infection, and HIV-1 Tat protein, activate K111 provirus expression in part by inducing loss of heterochromatin. HIV-1 infection of cell lines ( A ) or human peripheral blood lymphocytes (PBLs; B ) activates the expression of K111 as detected by qRT-PCR

    Techniques Used: Infection, Expressing, Quantitative RT-PCR

    Identification and genomic organization of K111 proviruses. ( A ) Quantitation of K111 env titers by qRT-PCR in the plasma of patients with HIV-1 and other diseases. The K111 env titers were measured by qRT-PCR using the probe K111P (see Supplemental Methods)
    Figure Legend Snippet: Identification and genomic organization of K111 proviruses. ( A ) Quantitation of K111 env titers by qRT-PCR in the plasma of patients with HIV-1 and other diseases. The K111 env titers were measured by qRT-PCR using the probe K111P (see Supplemental Methods)

    Techniques Used: Quantitation Assay, Quantitative RT-PCR

    Identification of K111 proviruses in individual human chromosomes. Bayesian inference tree of the 5′ LTR and 3′ LTRs, and flanking CER:D22Z3 sequences, of K111 proviruses amplified from human chromosomes. Sequences are colored to indicate
    Figure Legend Snippet: Identification of K111 proviruses in individual human chromosomes. Bayesian inference tree of the 5′ LTR and 3′ LTRs, and flanking CER:D22Z3 sequences, of K111 proviruses amplified from human chromosomes. Sequences are colored to indicate

    Techniques Used: Amplification

    ChIP analysis shows that K111 proviruses are found in centromeric and pericentromeric regions. Quantitative PCR of K111 DNA ( A ), and the centromeric 11-mer alphoid repeat of chromosome 21 (alphoid Chr.21 ) DNA ( B ), immunoprecipitated by antibodies to CENPA
    Figure Legend Snippet: ChIP analysis shows that K111 proviruses are found in centromeric and pericentromeric regions. Quantitative PCR of K111 DNA ( A ), and the centromeric 11-mer alphoid repeat of chromosome 21 (alphoid Chr.21 ) DNA ( B ), immunoprecipitated by antibodies to CENPA

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

    33) Product Images from "Molecular evolution in Panagrolaimus nematodes: origins of parthenogenesis, hermaphroditism and the Antarctic species P. davidi"

    Article Title: Molecular evolution in Panagrolaimus nematodes: origins of parthenogenesis, hermaphroditism and the Antarctic species P. davidi

    Journal: BMC Evolutionary Biology

    doi: 10.1186/1471-2148-9-15

    Lack of sperm and sperm nuclei from parthenogenetic Panagrolaimus strain JB115 . DIC imaging was used to search for sperm and epifluorescence to search for compact sperm nuclei (none were observed). See Methods for details. An egg is indicated by
    Figure Legend Snippet: Lack of sperm and sperm nuclei from parthenogenetic Panagrolaimus strain JB115 . DIC imaging was used to search for sperm and epifluorescence to search for compact sperm nuclei (none were observed). See Methods for details. An egg is indicated by "E" and the vulva is indicated by "V". The scalebar represents 40 μm.

    Techniques Used: Imaging

    Identification of sperm and sperm nuclei from hermaphroditic Panagrolaimus strain JU765 . DIC imaging was used to identify sperm (left, panels A and C) and epifluorescence imaging was used to identify characteristically compact sperm nuclei (right, panels B and D). See Methods for details. A and B show nematode midbodies; C and D show a dissected spermatheca. Ann egg is indicated by
    Figure Legend Snippet: Identification of sperm and sperm nuclei from hermaphroditic Panagrolaimus strain JU765 . DIC imaging was used to identify sperm (left, panels A and C) and epifluorescence imaging was used to identify characteristically compact sperm nuclei (right, panels B and D). See Methods for details. A and B show nematode midbodies; C and D show a dissected spermatheca. Ann egg is indicated by "E", sperm nuclei by "SN", oocytes by "O", and sperm by "S". The scalebar represents 40 μm.

    Techniques Used: Imaging

    Nuclear rRNA gene phylogeny for Panagrolaimus . Cladogram shown is a 80% bootstrap consensus tree for NJ analysis of aligned 18S and 28S rRNA sequences from Panagrolaimus and select outgroup nematode species and strains. The MP phylogeny was highly congruent with the NJ phylogeny shown – see Additional files 1 and 2 . Node-specific bootstrap values (1,000 replicates for each analysis) are shown, with NJ values over MP values. All strains and species historically considered as members of the Panagrolaimus genus are indicated by the brackets. Arrows denote the positions of two rhabditid strains initially misidentified as Panagrolaimus . Blue lines indicate parthenogenetic strains and green lines indicate hermaphroditic strains of Panagrolaimus . H. gingivalis also reproduces parthenogenetically in infected horses. DD13 and DD18 are two strains isolated from Newport, OR USA that are closely related to Rhabditophanes sp . KR3021. All other outgroup sequences were retrieved from Genbank.
    Figure Legend Snippet: Nuclear rRNA gene phylogeny for Panagrolaimus . Cladogram shown is a 80% bootstrap consensus tree for NJ analysis of aligned 18S and 28S rRNA sequences from Panagrolaimus and select outgroup nematode species and strains. The MP phylogeny was highly congruent with the NJ phylogeny shown – see Additional files 1 and 2 . Node-specific bootstrap values (1,000 replicates for each analysis) are shown, with NJ values over MP values. All strains and species historically considered as members of the Panagrolaimus genus are indicated by the brackets. Arrows denote the positions of two rhabditid strains initially misidentified as Panagrolaimus . Blue lines indicate parthenogenetic strains and green lines indicate hermaphroditic strains of Panagrolaimus . H. gingivalis also reproduces parthenogenetically in infected horses. DD13 and DD18 are two strains isolated from Newport, OR USA that are closely related to Rhabditophanes sp . KR3021. All other outgroup sequences were retrieved from Genbank.

    Techniques Used: Infection, Isolation

    Mitochondrial ND5 gene phylogeny for Panagrolaimus . Phylogram shows relationships among a subset of Panagrolaimus ND5 sequences (those able to be successfully amplified) inferred from NJ phylogenetic analysis (see Methods). Scale bar shows 0.05 substitutions per site. Bootstrap values resulting from NJ and MP analyses are shown for select nodes (e.g. those supporting PI, PIp and HI). Additional files 4 and 5 show complete phylograms with all bootstrap values for NJ and MP analyses, respectively. Blue lines indicate parthenogenetic strains and green lines indicate hermaphroditic strains.
    Figure Legend Snippet: Mitochondrial ND5 gene phylogeny for Panagrolaimus . Phylogram shows relationships among a subset of Panagrolaimus ND5 sequences (those able to be successfully amplified) inferred from NJ phylogenetic analysis (see Methods). Scale bar shows 0.05 substitutions per site. Bootstrap values resulting from NJ and MP analyses are shown for select nodes (e.g. those supporting PI, PIp and HI). Additional files 4 and 5 show complete phylograms with all bootstrap values for NJ and MP analyses, respectively. Blue lines indicate parthenogenetic strains and green lines indicate hermaphroditic strains.

    Techniques Used: Amplification

    34) 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

    35) Product Images from "ATLAS: A System to Selectively Identify Human-Specific L1 Insertions"

    Article Title: ATLAS: A System to Selectively Identify Human-Specific L1 Insertions

    Journal: American Journal of Human Genetics

    doi:

    ATLAS products' segregation in families. 5′ Ta-1d–specific ATLAS was performed on a three-generation CEPH pedigree (1331). FF = paternal grandfather; FM = paternal grandmother; F = father; S/D = son/daughter; M = mother; MF = maternal grandfather; MM = maternal grandmother. “C1” denotes a control sample in which water was substituted for genomic DNA at digestion; “C2” denotes a control sample in which water was substituted for library DNA; and “C3” and “C4” denote control samples in which water was substituted for the primary PCR products in the labeling reaction. Arrows (“A”–“D”) indicate dimorphic array products. MW = molecular weight markers.
    Figure Legend Snippet: ATLAS products' segregation in families. 5′ Ta-1d–specific ATLAS was performed on a three-generation CEPH pedigree (1331). FF = paternal grandfather; FM = paternal grandmother; F = father; S/D = son/daughter; M = mother; MF = maternal grandfather; MM = maternal grandmother. “C1” denotes a control sample in which water was substituted for genomic DNA at digestion; “C2” denotes a control sample in which water was substituted for library DNA; and “C3” and “C4” denote control samples in which water was substituted for the primary PCR products in the labeling reaction. Arrows (“A”–“D”) indicate dimorphic array products. MW = molecular weight markers.

    Techniques Used: Polymerase Chain Reaction, Labeling, Molecular Weight

    36) 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

    37) 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

    38) 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 ( Holloway et al., 2011 ). 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 ( Holloway et al., 2011 ). 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

    39) 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

    40) 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

    Related Articles

    Clone Assay:

    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
    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. .. Prior to electroporation constructs were linearized with ScaI.

    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).

    Amplification:

    Article Title: Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells
    Article Snippet: .. A BAC clone (bMQ-415O10; Geneservice) from 129S7/AB2.2 mouse DNA (indexed 129S7/SvEvBrd-Hprtb-m2 (AB2.2) library, displayed on the Ensembl Genome Browser [ http://www.ensembl.org/Mus_musculus/ ] under the DAS source 129S7/AB2.2) containing the USP3 locus was used to generate the USP3 targeting construct, and DNA fragments were amplified from the BAC DNA by PCR using the Expand Long range dNTPack kit (Roche). .. Fragments were as follows: the 5′ homology arm (3.5 kb PCR fragment 5′ of exon 2 in the USP3 gene, flanked by AscI restriction site: forward primer, 5′-GGCGCGCCCTGCCTCTGCTTCCTTTAGT-3′; reverse primer, 5′-GGCGCGCCGAATTCGGTTAATAAACTGGTTTCCCA-3′), the conditional arm (4.3 kb PCR product containing exon 2 and 3, flanked by PacI sites; forward primer containing PacI-BamHI-KpnI sites, 5′-TTAATTAAGGATCCGGTACCTGTTGACTTCAAAAGAGAGTG-3′; reverse primer, 5′-TTAATTAACCTCTTAAAGTCTAATACACTTC-3′), and the 3′ homology arm (4.6 kb PCR fragment 3′ of exon 3, flanked by NotI sites; forward primer, 5′-GCGGCCGCAAGTTGGAGATAGCCATGCC-3′; reverse primer, 5′-GCGGCCGCCTGTTAAAAGCAGATAAGCAC-3′).

    Article Title: Activation of the Farnesoid X Receptor Provides Protection against Acetaminophen-Induced Hepatic Toxicity
    Article Snippet: .. For ChIP-on-chip array hybridizations, mice were administered either vehicle or 50 mg/kg GW4064 dissolved in vehicle by oral gavage for 5 d. After ChIP, the FXR, IgG control, and input DNA for both control and GW4064-treated groups were prepared for hybridization to the 2.5-kb mouse promoter array (NimbleGen/Roche) using a random PCR amplification method according to the company’s protocol. .. The hybridization data were analyzed by using NimbleScan 2.3 and SignalMap software from NimbleGen/Roche.

    Construct:

    Article Title: Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells
    Article Snippet: .. A BAC clone (bMQ-415O10; Geneservice) from 129S7/AB2.2 mouse DNA (indexed 129S7/SvEvBrd-Hprtb-m2 (AB2.2) library, displayed on the Ensembl Genome Browser [ http://www.ensembl.org/Mus_musculus/ ] under the DAS source 129S7/AB2.2) containing the USP3 locus was used to generate the USP3 targeting construct, and DNA fragments were amplified from the BAC DNA by PCR using the Expand Long range dNTPack kit (Roche). .. Fragments were as follows: the 5′ homology arm (3.5 kb PCR fragment 5′ of exon 2 in the USP3 gene, flanked by AscI restriction site: forward primer, 5′-GGCGCGCCCTGCCTCTGCTTCCTTTAGT-3′; reverse primer, 5′-GGCGCGCCGAATTCGGTTAATAAACTGGTTTCCCA-3′), the conditional arm (4.3 kb PCR product containing exon 2 and 3, flanked by PacI sites; forward primer containing PacI-BamHI-KpnI sites, 5′-TTAATTAAGGATCCGGTACCTGTTGACTTCAAAAGAGAGTG-3′; reverse primer, 5′-TTAATTAACCTCTTAAAGTCTAATACACTTC-3′), and the 3′ homology arm (4.6 kb PCR fragment 3′ of exon 3, flanked by NotI sites; forward primer, 5′-GCGGCCGCAAGTTGGAGATAGCCATGCC-3′; reverse primer, 5′-GCGGCCGCCTGTTAAAAGCAGATAAGCAC-3′).

    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
    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. .. Prior to electroporation constructs were linearized with ScaI.

    Mouse Assay:

    Article Title: Activation of the Farnesoid X Receptor Provides Protection against Acetaminophen-Induced Hepatic Toxicity
    Article Snippet: .. For ChIP-on-chip array hybridizations, mice were administered either vehicle or 50 mg/kg GW4064 dissolved in vehicle by oral gavage for 5 d. After ChIP, the FXR, IgG control, and input DNA for both control and GW4064-treated groups were prepared for hybridization to the 2.5-kb mouse promoter array (NimbleGen/Roche) using a random PCR amplification method according to the company’s protocol. .. The hybridization data were analyzed by using NimbleScan 2.3 and SignalMap software from NimbleGen/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.

    Article Title: Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells
    Article Snippet: .. A BAC clone (bMQ-415O10; Geneservice) from 129S7/AB2.2 mouse DNA (indexed 129S7/SvEvBrd-Hprtb-m2 (AB2.2) library, displayed on the Ensembl Genome Browser [ http://www.ensembl.org/Mus_musculus/ ] under the DAS source 129S7/AB2.2) containing the USP3 locus was used to generate the USP3 targeting construct, and DNA fragments were amplified from the BAC DNA by PCR using the Expand Long range dNTPack kit (Roche). .. Fragments were as follows: the 5′ homology arm (3.5 kb PCR fragment 5′ of exon 2 in the USP3 gene, flanked by AscI restriction site: forward primer, 5′-GGCGCGCCCTGCCTCTGCTTCCTTTAGT-3′; reverse primer, 5′-GGCGCGCCGAATTCGGTTAATAAACTGGTTTCCCA-3′), the conditional arm (4.3 kb PCR product containing exon 2 and 3, flanked by PacI sites; forward primer containing PacI-BamHI-KpnI sites, 5′-TTAATTAAGGATCCGGTACCTGTTGACTTCAAAAGAGAGTG-3′; reverse primer, 5′-TTAATTAACCTCTTAAAGTCTAATACACTTC-3′), and the 3′ homology arm (4.6 kb PCR fragment 3′ of exon 3, flanked by NotI sites; forward primer, 5′-GCGGCCGCAAGTTGGAGATAGCCATGCC-3′; reverse primer, 5′-GCGGCCGCCTGTTAAAAGCAGATAAGCAC-3′).

    Article Title: Effect of Leflunomide, Cidofovir and Ciprofloxacin on Replication of BKPyV in a Salivary Gland In Vitro Culture System
    Article Snippet: .. BKPyV VP1 gene forward (BKPVWGF; 5’-GCGGGATCCAGATGAAAACCTTAGG-3’) and reverse primers (BKPyVWGR; 5’-GCGGGATCCCCCATTTCTGG-3’) including the naturally occurring BamH1 restriction fragment recognition sites were used to amplify the whole genome (wg) of BKPyV via PCR from throat wash of HIVSGD patients, and the urine of a lung transplant patient using the Expand Long Range dNTPack (Roche) as described by manufacturer. .. Amplified wg BKPyV products were purified by QIAquick PCR Purification Kit (QIAGEN) as described by manufacturer.

    Article Title: Activation of the Farnesoid X Receptor Provides Protection against Acetaminophen-Induced Hepatic Toxicity
    Article Snippet: .. For ChIP-on-chip array hybridizations, mice were administered either vehicle or 50 mg/kg GW4064 dissolved in vehicle by oral gavage for 5 d. After ChIP, the FXR, IgG control, and input DNA for both control and GW4064-treated groups were prepared for hybridization to the 2.5-kb mouse promoter array (NimbleGen/Roche) using a random PCR amplification method according to the company’s protocol. .. The hybridization data were analyzed by using NimbleScan 2.3 and SignalMap software from NimbleGen/Roche.

    Article Title: HiDRA-seq: High-Throughput SARS-CoV-2 Detection by RNA Barcoding and Amplicon Sequencing
    Article Snippet: .. Each PCR reaction was assembled by combining, in individual tubes, 5 ul of the three amplicons with 20 μl of PCR master mix containing 1.25X KAPA HiFi HotStart ReadyMix (Roche), 0.375 uM of the i5 primer (Microsynth AG) and 0.375 uM of the barcoded i7 primer (Illumina). ..

    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
    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. .. Prior to electroporation constructs were linearized with ScaI.

    BAC Assay:

    Article Title: Tight regulation of ubiquitin-mediated DNA damage response by USP3 preserves the functional integrity of hematopoietic stem cells
    Article Snippet: .. A BAC clone (bMQ-415O10; Geneservice) from 129S7/AB2.2 mouse DNA (indexed 129S7/SvEvBrd-Hprtb-m2 (AB2.2) library, displayed on the Ensembl Genome Browser [ http://www.ensembl.org/Mus_musculus/ ] under the DAS source 129S7/AB2.2) containing the USP3 locus was used to generate the USP3 targeting construct, and DNA fragments were amplified from the BAC DNA by PCR using the Expand Long range dNTPack kit (Roche). .. Fragments were as follows: the 5′ homology arm (3.5 kb PCR fragment 5′ of exon 2 in the USP3 gene, flanked by AscI restriction site: forward primer, 5′-GGCGCGCCCTGCCTCTGCTTCCTTTAGT-3′; reverse primer, 5′-GGCGCGCCGAATTCGGTTAATAAACTGGTTTCCCA-3′), the conditional arm (4.3 kb PCR product containing exon 2 and 3, flanked by PacI sites; forward primer containing PacI-BamHI-KpnI sites, 5′-TTAATTAAGGATCCGGTACCTGTTGACTTCAAAAGAGAGTG-3′; reverse primer, 5′-TTAATTAACCTCTTAAAGTCTAATACACTTC-3′), and the 3′ homology arm (4.6 kb PCR fragment 3′ of exon 3, flanked by NotI sites; forward primer, 5′-GCGGCCGCAAGTTGGAGATAGCCATGCC-3′; reverse primer, 5′-GCGGCCGCCTGTTAAAAGCAGATAAGCAC-3′).

    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).

    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.

    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.

    Article Title: Activation of the Farnesoid X Receptor Provides Protection against Acetaminophen-Induced Hepatic Toxicity
    Article Snippet: .. For ChIP-on-chip array hybridizations, mice were administered either vehicle or 50 mg/kg GW4064 dissolved in vehicle by oral gavage for 5 d. After ChIP, the FXR, IgG control, and input DNA for both control and GW4064-treated groups were prepared for hybridization to the 2.5-kb mouse promoter array (NimbleGen/Roche) using a random PCR amplification method according to the company’s protocol. .. The hybridization data were analyzed by using NimbleScan 2.3 and SignalMap software from NimbleGen/Roche.

    Derivative Assay:

    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
    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. .. Prior to electroporation constructs were linearized with ScaI.

    Hybridization:

    Article Title: Activation of the Farnesoid X Receptor Provides Protection against Acetaminophen-Induced Hepatic Toxicity
    Article Snippet: .. For ChIP-on-chip array hybridizations, mice were administered either vehicle or 50 mg/kg GW4064 dissolved in vehicle by oral gavage for 5 d. After ChIP, the FXR, IgG control, and input DNA for both control and GW4064-treated groups were prepared for hybridization to the 2.5-kb mouse promoter array (NimbleGen/Roche) using a random PCR amplification method according to the company’s protocol. .. The hybridization data were analyzed by using NimbleScan 2.3 and SignalMap software from NimbleGen/Roche.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 85
    Roche long range pcr expand long pcr kit roche
    LCT13 and <t>TFPI-2as</t> expression is linked. ( A ) Schematic diagram of the genomic region in Figure 1 A indicating regions (1–7) analysed by strand-specific <t>RT–PCR</t> (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).
    Long Range Pcr Expand Long Pcr Kit Roche, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/long range pcr expand long pcr kit roche/product/Roche
    Average 85 stars, based on 62 article reviews
    Price from $9.99 to $1999.99
    long range pcr expand long pcr kit roche - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    89
    Roche long range pcr kit
    IL-1β induction and chromatin structure of cPLA 2 <t>α</t> in HFL-1. (A, left) HFL-1 cells were treated with 2 ng/µl IL-1β for the indicated times, and total RNA was analyzed by real-time <t>RT-PCR.</t> Relative fold induction was calculated
    Long Range Pcr Kit, supplied by Roche, used in various techniques. Bioz Stars score: 89/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/long range pcr kit/product/Roche
    Average 89 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    long range pcr kit - by Bioz Stars, 2020-09
    89/100 stars
      Buy from Supplier

    Image Search Results


    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

    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).)

    Journal: BioMed Research International

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

    doi: 10.1155/2014/256245

    Figure Lengend 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).)

    Article Snippet: PCR Long-Range On absence of amplification of POLH exon 10, long PCR was performed using the Expand Long Template PCR System Kit (Expand Long Range dNTPack 700 units/μ L Roche).

    Techniques: Polymerase Chain Reaction, Marker

    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.

    Journal: BMC Evolutionary Biology

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

    doi: 10.1186/1471-2148-8-62

    Figure Lengend 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.

    Article Snippet: PCR, DNA sequencing and phylogenetics mtDNA sequencing and PCR were performed as previously described [ , ], with the exception that mitochondrial genome sequences were initially amplified as four overlapping PCR products 3–5 kb in size each using the Expand Long Range PCR kit (Roche). e2TAK (Takara) proofreading DNA polymerase was used for all conventional PCRs.

    Techniques: Polymerase Chain Reaction

    IL-1β induction and chromatin structure of cPLA 2 α in HFL-1. (A, left) HFL-1 cells were treated with 2 ng/µl IL-1β for the indicated times, and total RNA was analyzed by real-time RT-PCR. Relative fold induction was calculated

    Journal: Journal of Lipid Research

    Article Title: A distal enhancer controls cytokine-dependent human cPLA2? gene expression [S]

    doi: 10.1194/jlr.M037382

    Figure Lengend Snippet: IL-1β induction and chromatin structure of cPLA 2 α in HFL-1. (A, left) HFL-1 cells were treated with 2 ng/µl IL-1β for the indicated times, and total RNA was analyzed by real-time RT-PCR. Relative fold induction was calculated

    Article Snippet: The −14, −5.5, and −4.1 kb cPLA2 α promoters were amplified from using the Long Range PCR kit (Roche), with the forward primers 5′-AGAGTTGGGATGGAGAAGGTTG-3′, 5′-TGCAAAGTGCCTGCCAGTC-3′, or 5′-GATGGAGATGGCAGTGGCAG-3′, respectively, and the reverse primer 5′-GCTTACAGTTCCCAGAGTTACC-3′, and cloned into the TOPO-XL vector.

    Techniques: Quantitative RT-PCR