homology directed repair kpn i hf  (New England Biolabs)


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    KpnI HF
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    KpnI HF 20 000 units
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    r3142l
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    Restriction Enzymes
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    New England Biolabs homology directed repair kpn i hf
    KpnI HF
    KpnI HF 20 000 units
    https://www.bioz.com/result/homology directed repair kpn i hf/product/New England Biolabs
    Average 98 stars, based on 17907 article reviews
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    homology directed repair kpn i hf - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair"

    Article Title: Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36506-w

    CRISPR/Cas9 templated gene editing analysis in mouse fibroblasts. ( A ) Overview of the sgRNA + 1 target site with sections of the sense and antisense (AS) ssODN that remove the c.828TAAA insertion and introduce a Kpn I recognition site by a silent C > A substitution. Predicted cut site for Cas9 are indicated by “ ˄ ” and “ ”, with “ ” indicating the strand that is nicked by Cas9D10A. Nucleotide numbers refer to the mutant c.828InsTAAA Fancf gene. ( B ) Overall gene editing efficiencies obtained in mouse fibroblasts depicted as general indel frequencies determined by TIDER (n = 3, one sided Student’s T-test, *p = 0.02). ( C ) Restriction fragment length polymorphism (RFLP) analysis by Kpn I digest of the Fancf locus provides evidence for template-based repair after Cas9, sgRNA + 1 and ssODN transfection (n = 3, one sided Student’s T-test, *p
    Figure Legend Snippet: CRISPR/Cas9 templated gene editing analysis in mouse fibroblasts. ( A ) Overview of the sgRNA + 1 target site with sections of the sense and antisense (AS) ssODN that remove the c.828TAAA insertion and introduce a Kpn I recognition site by a silent C > A substitution. Predicted cut site for Cas9 are indicated by “ ˄ ” and “ ”, with “ ” indicating the strand that is nicked by Cas9D10A. Nucleotide numbers refer to the mutant c.828InsTAAA Fancf gene. ( B ) Overall gene editing efficiencies obtained in mouse fibroblasts depicted as general indel frequencies determined by TIDER (n = 3, one sided Student’s T-test, *p = 0.02). ( C ) Restriction fragment length polymorphism (RFLP) analysis by Kpn I digest of the Fancf locus provides evidence for template-based repair after Cas9, sgRNA + 1 and ssODN transfection (n = 3, one sided Student’s T-test, *p

    Techniques Used: CRISPR, Introduce, Mutagenesis, Transfection

    2) Product Images from "IQGAP1 Negatively Regulates HIV-1 Gag Trafficking and Virion Production"

    Article Title: IQGAP1 Negatively Regulates HIV-1 Gag Trafficking and Virion Production

    Journal: Cell reports

    doi: 10.1016/j.celrep.2020.03.002

    IQGAP1 Interacts with the Nucleocapsid and p6 Domains of HIV-1 Gag (A) IQGAP1 interacts with the HIV-1 Gag protein. HEK293T cells were co-transfected with HIV-1 viral genome (pNL4–3.Luc.R-E-; 1 µg) together with DNAs expressing GFP or GFP-IQGAP1 at a molar ratio of 3 (GFP or GFP-IQGAP1 to pNL4–3.Luc.R-E-). Cell lysates were subjected to immunoprecipitation using anti-GFP antibody. GFP, GFP-IQGAP1, and Gag were detected using western blot. Shown are representative western blots from three independent experiments (n = 3). (B) IQGAP1 interacts with unmyristylated HIV-1 Gag protein. HEK293T cells were co-transfected with HIV-1 viral genome (Gag WT; 1 µg) or with an HIV-1 genome encoding a Gag mutant that cannot be myristylated (Gag G2A; 1 µg) together with DNAs encoding GFP or GFP-IQGAP1 at a molar ratio of 3 (GFP or GFP-IQGAP1 to Gag WT or Gag G2A) and processed as in (A). Shown are representative western blots from three independent experiments (n = 3). (C) Schematic presentation of the Rev-independent flag-tagged Gag domains proteins used for this experiment. (D) The nucleocapsid domain of Gag interacts with IQGAP1. HEK293T cells were co-transfected with DNAs encoding GFP or GFP-IQGAP1 together with 1 µg of the indicated Gag constructs from (C) at a molar ratio of 3 (GFP or GFP-IQGAP1 to HIV-1 Gag constructs) and processed as in (A). Shown are representative western blots from three independent experiments (n = 3). (E) The p6 domain of Gag interacts with IQGAP1. HEK293T cells were co-transfected with the GagDNC construct (C) (1 µg) together with DNAs encoding GFP or GFP-IQGAP1 at a molar ratio of 3 (GFP or GFP-IQGAP1 to GagΔDNC) and processed as in (A). Shown are representative western blots from three independent experiments (n = 3).
    Figure Legend Snippet: IQGAP1 Interacts with the Nucleocapsid and p6 Domains of HIV-1 Gag (A) IQGAP1 interacts with the HIV-1 Gag protein. HEK293T cells were co-transfected with HIV-1 viral genome (pNL4–3.Luc.R-E-; 1 µg) together with DNAs expressing GFP or GFP-IQGAP1 at a molar ratio of 3 (GFP or GFP-IQGAP1 to pNL4–3.Luc.R-E-). Cell lysates were subjected to immunoprecipitation using anti-GFP antibody. GFP, GFP-IQGAP1, and Gag were detected using western blot. Shown are representative western blots from three independent experiments (n = 3). (B) IQGAP1 interacts with unmyristylated HIV-1 Gag protein. HEK293T cells were co-transfected with HIV-1 viral genome (Gag WT; 1 µg) or with an HIV-1 genome encoding a Gag mutant that cannot be myristylated (Gag G2A; 1 µg) together with DNAs encoding GFP or GFP-IQGAP1 at a molar ratio of 3 (GFP or GFP-IQGAP1 to Gag WT or Gag G2A) and processed as in (A). Shown are representative western blots from three independent experiments (n = 3). (C) Schematic presentation of the Rev-independent flag-tagged Gag domains proteins used for this experiment. (D) The nucleocapsid domain of Gag interacts with IQGAP1. HEK293T cells were co-transfected with DNAs encoding GFP or GFP-IQGAP1 together with 1 µg of the indicated Gag constructs from (C) at a molar ratio of 3 (GFP or GFP-IQGAP1 to HIV-1 Gag constructs) and processed as in (A). Shown are representative western blots from three independent experiments (n = 3). (E) The p6 domain of Gag interacts with IQGAP1. HEK293T cells were co-transfected with the GagDNC construct (C) (1 µg) together with DNAs encoding GFP or GFP-IQGAP1 at a molar ratio of 3 (GFP or GFP-IQGAP1 to GagΔDNC) and processed as in (A). Shown are representative western blots from three independent experiments (n = 3).

    Techniques Used: Transfection, Expressing, Immunoprecipitation, Western Blot, Mutagenesis, Construct

    The C Term inus Region of HIV-1 Gag Is Required for IQGAP1 Regulation of Viral Release Independently of Gag L Domains (A) Schematic presentation of Rev-independent flag-tagged Gag domains proteins used for this experiment. (B and C) Replacement of the C terminus region of Gag with the GCN4 leucine zipper abolishes IQGAP1 regulation of HIV-1 VLP release. HEK293T cells were co-transfected with Rev-independent Gag-flag (B) or GagZ-flag (C) constructs (0.5 µg) with increasing amounts of DNA encoding GFP-IQGAP1 or GFP at a molar ratio (GFP-IQGAP1 to HIV-1 Gag DNAs) ranging from 2 to 6 and an empty vector to ensure equal total amount of transfected DNAs. After 48 h, cells were lysed and virions in the culture supernatants were purified. CA levels in pelleted virions and intracellular GFP-IQGAP1 and Gag levels were assessed using western blot with specific anti-GFP or anti-flag antibodies. Shown are representative western blots from three independent experiments (n = 3). (D and E) Either the p6 or the nucleocapsid domains in Gag are sufficient for IQGAP1 negative regulation of HIV-1 VLP release. HEK293T cells were co-transfected with Rev-independent GagΔp6-flag (D) or GagDNC-flag (E) DNAs (0.5 µg) together with increasing amounts of DNAs encoding GFP or GFP-IQGAP1 at a molar ratio (GFP-IQGAP1 to HIV-1 Gag DNAs) ranging from 2 to 6 and an empty vector to ensure equal total amount of transfected DNA and processed as in (B). Shown are representative western blots from three independent experiments (n = 3). (F) Schematic presentation of Rev-independent flag-tagged Gag mutant proteins used for this research. (G–J) IQGAP1 overexpression reduces HIV-1 Gag VLPs lacking the ability to recruit the cellular ESCRT machinery. HEK293T cells were co-transfected with DNA constructs from (F) (0.5 µg) together with increasing amounts of DNAs encoding GFP or GFP-IQGAP1 at a molar ratio (GFP-IQGAP1 to HIV-1 Gag DNAs) ranging from 2 to 6 and an empty vector to ensure equal total amount of transfected DNAs. After 48 h, cells were lysed and virions in the supernatants were isolated. CA levels in pelleted virions and cellular GFP-IQGAP1 and Gag expression levels were assessed using western blot with specific anti-GFP or anti-flag antibodies. Cells were transfected with (G) wild-type, (H) PTAPmut, (I) LYPXnLmut, (J) and PTAP/LYPXnLmut Gag expression constructs. Shown are representative western blots from three independent experiments (n = 3).
    Figure Legend Snippet: The C Term inus Region of HIV-1 Gag Is Required for IQGAP1 Regulation of Viral Release Independently of Gag L Domains (A) Schematic presentation of Rev-independent flag-tagged Gag domains proteins used for this experiment. (B and C) Replacement of the C terminus region of Gag with the GCN4 leucine zipper abolishes IQGAP1 regulation of HIV-1 VLP release. HEK293T cells were co-transfected with Rev-independent Gag-flag (B) or GagZ-flag (C) constructs (0.5 µg) with increasing amounts of DNA encoding GFP-IQGAP1 or GFP at a molar ratio (GFP-IQGAP1 to HIV-1 Gag DNAs) ranging from 2 to 6 and an empty vector to ensure equal total amount of transfected DNAs. After 48 h, cells were lysed and virions in the culture supernatants were purified. CA levels in pelleted virions and intracellular GFP-IQGAP1 and Gag levels were assessed using western blot with specific anti-GFP or anti-flag antibodies. Shown are representative western blots from three independent experiments (n = 3). (D and E) Either the p6 or the nucleocapsid domains in Gag are sufficient for IQGAP1 negative regulation of HIV-1 VLP release. HEK293T cells were co-transfected with Rev-independent GagΔp6-flag (D) or GagDNC-flag (E) DNAs (0.5 µg) together with increasing amounts of DNAs encoding GFP or GFP-IQGAP1 at a molar ratio (GFP-IQGAP1 to HIV-1 Gag DNAs) ranging from 2 to 6 and an empty vector to ensure equal total amount of transfected DNA and processed as in (B). Shown are representative western blots from three independent experiments (n = 3). (F) Schematic presentation of Rev-independent flag-tagged Gag mutant proteins used for this research. (G–J) IQGAP1 overexpression reduces HIV-1 Gag VLPs lacking the ability to recruit the cellular ESCRT machinery. HEK293T cells were co-transfected with DNA constructs from (F) (0.5 µg) together with increasing amounts of DNAs encoding GFP or GFP-IQGAP1 at a molar ratio (GFP-IQGAP1 to HIV-1 Gag DNAs) ranging from 2 to 6 and an empty vector to ensure equal total amount of transfected DNAs. After 48 h, cells were lysed and virions in the supernatants were isolated. CA levels in pelleted virions and cellular GFP-IQGAP1 and Gag expression levels were assessed using western blot with specific anti-GFP or anti-flag antibodies. Cells were transfected with (G) wild-type, (H) PTAPmut, (I) LYPXnLmut, (J) and PTAP/LYPXnLmut Gag expression constructs. Shown are representative western blots from three independent experiments (n = 3).

    Techniques Used: Transfection, Construct, Plasmid Preparation, Purification, Western Blot, Mutagenesis, Over Expression, Isolation, Expressing

    3) Product Images from "The ter Mutation in the Rat Dnd1 Gene Initiates Gonadal Teratomas and Infertility in Both Genders"

    Article Title: The ter Mutation in the Rat Dnd1 Gene Initiates Gonadal Teratomas and Infertility in Both Genders

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0038001

    Genotyping, survival and tumor progression. ( A ) Genotyping. The ter mutation disrupts a Kpn I restriction site used for genotyping of the ter allele performing PCR amplification and restriction digest. Kpn I digest (black arrows) cleaved the PCR product of 560 bp into 380 bp and 180 bp fragments, N negative control. ( B ) Kaplan-Meyer survival analysis of male and female WKY- Dnd1 ter /Ztm rats carrying heterozygous and homozygous ter alleles compared to wild type animals. ( C ) Sizes of TGCTs and OGCTs after 3, 6 and 9 weeks of age in homozygous WKY- Dnd1 ter /Ztm rats.
    Figure Legend Snippet: Genotyping, survival and tumor progression. ( A ) Genotyping. The ter mutation disrupts a Kpn I restriction site used for genotyping of the ter allele performing PCR amplification and restriction digest. Kpn I digest (black arrows) cleaved the PCR product of 560 bp into 380 bp and 180 bp fragments, N negative control. ( B ) Kaplan-Meyer survival analysis of male and female WKY- Dnd1 ter /Ztm rats carrying heterozygous and homozygous ter alleles compared to wild type animals. ( C ) Sizes of TGCTs and OGCTs after 3, 6 and 9 weeks of age in homozygous WKY- Dnd1 ter /Ztm rats.

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

    4) Product Images from "Hypoxia-NOTCH1-SOX2 signaling is important for maintaining cancer stem cells in ovarian cancer"

    Article Title: Hypoxia-NOTCH1-SOX2 signaling is important for maintaining cancer stem cells in ovarian cancer

    Journal: Oncotarget

    doi: 10.18632/oncotarget.10954

    Hypoxia and NOTCH1 signaling increase SOX2 promoter activity A. SOX promoter activity in adherent A2780 cells after CoCl 2 treatment (100 μM, 48 h) is shown. Data indicate mean ± SD (n=4). *, P
    Figure Legend Snippet: Hypoxia and NOTCH1 signaling increase SOX2 promoter activity A. SOX promoter activity in adherent A2780 cells after CoCl 2 treatment (100 μM, 48 h) is shown. Data indicate mean ± SD (n=4). *, P

    Techniques Used: Activity Assay

    NOTCH1 and SOX2 are important for maintaining CSC properties in primary ovarian cancer cells A. RT-PCR results of spheres generated from primary epithelial ovarian cancer cells are shown with indicated probes. B. Confocal microscopy images of spheres generated from EOC-12 (left panels) and EOC-15 (right panels) after immunolabeling with anti-SOX2 antibody. Nuclei were stained with DAPI. Scale bar = 100 μm. C. Schematic outline of the sphere enrichment protocol for primary EOC culture (100 μm
    Figure Legend Snippet: NOTCH1 and SOX2 are important for maintaining CSC properties in primary ovarian cancer cells A. RT-PCR results of spheres generated from primary epithelial ovarian cancer cells are shown with indicated probes. B. Confocal microscopy images of spheres generated from EOC-12 (left panels) and EOC-15 (right panels) after immunolabeling with anti-SOX2 antibody. Nuclei were stained with DAPI. Scale bar = 100 μm. C. Schematic outline of the sphere enrichment protocol for primary EOC culture (100 μm

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Generated, Confocal Microscopy, Immunolabeling, Staining

    SOX2 expression is important for maintaining chemoresistance in ovarian cancer cells A. Viability of adherent cells (AD) or sphere cells (SP) of A2780 (upper panel) or SKOV3 (lower panel) ovarian cancer cells in the presence of increasing concentrations of paclitaxel was determined by MTT assay. The percentage of viable cells is shown after normalization to no treatment control. Data indicate mean ± SD (n=4). *, P
    Figure Legend Snippet: SOX2 expression is important for maintaining chemoresistance in ovarian cancer cells A. Viability of adherent cells (AD) or sphere cells (SP) of A2780 (upper panel) or SKOV3 (lower panel) ovarian cancer cells in the presence of increasing concentrations of paclitaxel was determined by MTT assay. The percentage of viable cells is shown after normalization to no treatment control. Data indicate mean ± SD (n=4). *, P

    Techniques Used: Expressing, MTT Assay

    NOTCH1 and SOX2 are important for maintaining CSC properties in ovarian cancer cells A. Numbers of spheres generated from adherent A2780 cells with or without CoCl 2 (100 μM) treatment in combination with SOX2 knockdown are shown. The numbers of spheres were counted on day 7 after treatment. Data indicate mean ± SD (n=4). *, P
    Figure Legend Snippet: NOTCH1 and SOX2 are important for maintaining CSC properties in ovarian cancer cells A. Numbers of spheres generated from adherent A2780 cells with or without CoCl 2 (100 μM) treatment in combination with SOX2 knockdown are shown. The numbers of spheres were counted on day 7 after treatment. Data indicate mean ± SD (n=4). *, P

    Techniques Used: Generated

    SOX2 expression is increased in spheres of ovarian cancer cells A. Spheres were generated from confluent culture of adherent SKOV3, PA-1, and A2780 cells (upper panels) and maintained in suspension culture (lower panels). Spheres were photographed using an inverted microscope on day 7 after individual sphere cells were seeded into low attachment 6-well plates. Scale bar = 100 μm. B. RT-PCR results of adherent (AD) and sphere cells (SP) with indicated probes are shown. C. RT-PCR results of A2780-SP and SKOV3-SP cells with or without SOX2 knockdown are shown with indicated probes. D. Representative images of spheres generated from A2780-SP cells with or without SOX2 knockdown are shown. Scale bar = 100 μm. E. Numbers of spheres generated from A2780-SP or SKOV3-SP cells with or without SOX2 knockdown are shown. Data indicate mean ± SD (n=4). *, P
    Figure Legend Snippet: SOX2 expression is increased in spheres of ovarian cancer cells A. Spheres were generated from confluent culture of adherent SKOV3, PA-1, and A2780 cells (upper panels) and maintained in suspension culture (lower panels). Spheres were photographed using an inverted microscope on day 7 after individual sphere cells were seeded into low attachment 6-well plates. Scale bar = 100 μm. B. RT-PCR results of adherent (AD) and sphere cells (SP) with indicated probes are shown. C. RT-PCR results of A2780-SP and SKOV3-SP cells with or without SOX2 knockdown are shown with indicated probes. D. Representative images of spheres generated from A2780-SP cells with or without SOX2 knockdown are shown. Scale bar = 100 μm. E. Numbers of spheres generated from A2780-SP or SKOV3-SP cells with or without SOX2 knockdown are shown. Data indicate mean ± SD (n=4). *, P

    Techniques Used: Expressing, Generated, Inverted Microscopy, Reverse Transcription Polymerase Chain Reaction

    5) Product Images from "FOXP1 functions as an oncogene in promoting cancer stem cell-like characteristics in ovarian cancer cells"

    Article Title: FOXP1 functions as an oncogene in promoting cancer stem cell-like characteristics in ovarian cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.6510

    FOXP1 promotes the resistance of ovarian cancer cells to chemotherapeutic drugs A. The viability of A2780 ovarian cancer cells with or without FOXP1 knockdown (shFOXP1) or overexpression (FOXP1) was measured by MTT assay after treatment of cells with indicated concentrations of Paclitaxel (left panel) or Cisplatin (right panel). B. The viability of SKOV3 ovarian cancer cells with or without FOXP1 knockdown or overexpression was measured by MTT assay after treatment of cells with indicated concentrations of Paclitaxel (left panel) or Cisplatin (right panel). Data are presented as mean ± SD. *, p
    Figure Legend Snippet: FOXP1 promotes the resistance of ovarian cancer cells to chemotherapeutic drugs A. The viability of A2780 ovarian cancer cells with or without FOXP1 knockdown (shFOXP1) or overexpression (FOXP1) was measured by MTT assay after treatment of cells with indicated concentrations of Paclitaxel (left panel) or Cisplatin (right panel). B. The viability of SKOV3 ovarian cancer cells with or without FOXP1 knockdown or overexpression was measured by MTT assay after treatment of cells with indicated concentrations of Paclitaxel (left panel) or Cisplatin (right panel). Data are presented as mean ± SD. *, p

    Techniques Used: Over Expression, MTT Assay

    FOXP1 knockdown inhibits tumor growth in xenotransplantation of A2780 ovarian cancer cells A. Representative pictures of mice on day 35 after injection of A2780 ovarian cancer cells with or without FOXP1 knockdown are shown. Arrows indicate the injection sites. B, C. Representative pictures (B) and the average weight (C) of tumors removed from mice on day 35 after injection of A2780 ovarian cancer cells with or without FOXP1 knockdown into nude mice are shown. D. Measurements of tumor volume from day 14 to day 35 after injection of A2780 ovarian cancer cells with or without FOXP1 knockdown into nude mice are shown. Data are presented as mean ± SD ( n = 6).
    Figure Legend Snippet: FOXP1 knockdown inhibits tumor growth in xenotransplantation of A2780 ovarian cancer cells A. Representative pictures of mice on day 35 after injection of A2780 ovarian cancer cells with or without FOXP1 knockdown are shown. Arrows indicate the injection sites. B, C. Representative pictures (B) and the average weight (C) of tumors removed from mice on day 35 after injection of A2780 ovarian cancer cells with or without FOXP1 knockdown into nude mice are shown. D. Measurements of tumor volume from day 14 to day 35 after injection of A2780 ovarian cancer cells with or without FOXP1 knockdown into nude mice are shown. Data are presented as mean ± SD ( n = 6).

    Techniques Used: Mouse Assay, Injection

    FOXP1 promotes the spheroid formation of A2780 ovarian cancer cells A. Bright field images of spheroids generated from A2780 ovarian cancer cells with or without FOXP1 knockdown (shFOXP1) or overexpression (FOXP1) are shown from day 5 to day 20 of spheroid culture (bar = 100 μm). B. The size of spheroids generated from A2780 ovarian cancer cells with or without FOXP1 knockdown or overexpression was measured from day 5 to day 20 of spheroid culture. C. The number of spheroids generated from 1000 cells of A2780 ovarian cancer cells with or without FOXP1 knockdown or overexpression is shown from day 5 to day 20 of spheroid culture. D. The number of cells per spheroid generated from A2780 ovarian cancer cells with or without FOXP1 knockdown or overexpression is shown from day 5 to day 20 of spheroid culture. Data are presented as mean ± SD. *, p
    Figure Legend Snippet: FOXP1 promotes the spheroid formation of A2780 ovarian cancer cells A. Bright field images of spheroids generated from A2780 ovarian cancer cells with or without FOXP1 knockdown (shFOXP1) or overexpression (FOXP1) are shown from day 5 to day 20 of spheroid culture (bar = 100 μm). B. The size of spheroids generated from A2780 ovarian cancer cells with or without FOXP1 knockdown or overexpression was measured from day 5 to day 20 of spheroid culture. C. The number of spheroids generated from 1000 cells of A2780 ovarian cancer cells with or without FOXP1 knockdown or overexpression is shown from day 5 to day 20 of spheroid culture. D. The number of cells per spheroid generated from A2780 ovarian cancer cells with or without FOXP1 knockdown or overexpression is shown from day 5 to day 20 of spheroid culture. Data are presented as mean ± SD. *, p

    Techniques Used: Generated, Over Expression

    FOXP1 promotes expression of stemness-related genes and EMT-related genes RT-PCR analysis of A2780 ovarian cancer cells with or without FOXP1 knockdown (shFOXP1) or overexpression (FOXP1) was performed using probes for stemness-related genes A. or EMT-related genes B.
    Figure Legend Snippet: FOXP1 promotes expression of stemness-related genes and EMT-related genes RT-PCR analysis of A2780 ovarian cancer cells with or without FOXP1 knockdown (shFOXP1) or overexpression (FOXP1) was performed using probes for stemness-related genes A. or EMT-related genes B.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Over Expression

    FOXP1 promotes proliferation and migration of A2780 ovarian cancer cells A. Cell proliferation was measured by counting cells every day for four days after plating the same number (1×10 4 /well in 12-well culture plate) of A2780 ovarian cancer cells with or without FOXP1 knockdown (shFOXP1) or overexpression (FOXP1). B, C. Migration of A2780 ovarian cancer cells with or without FOXP1 knockdown or overexpression was measured by scratch wound healing assay. Bright field images (B) and quantification of wound gap (C) at 24 h, 48 h, and 72 h after application of scratch wound are shown. Wound gap was expressed as a percentage of initial wound gap. D, E. Migration of A2780 ovarian cancer cells with or without FOXP1 knockdown or overexpression was measured by transwell migration assay. Fluorescence microscope images of the cell migration (bar = 100 μm) (D) and quantification of migrated cells (E) at 12 h are shown.
    Figure Legend Snippet: FOXP1 promotes proliferation and migration of A2780 ovarian cancer cells A. Cell proliferation was measured by counting cells every day for four days after plating the same number (1×10 4 /well in 12-well culture plate) of A2780 ovarian cancer cells with or without FOXP1 knockdown (shFOXP1) or overexpression (FOXP1). B, C. Migration of A2780 ovarian cancer cells with or without FOXP1 knockdown or overexpression was measured by scratch wound healing assay. Bright field images (B) and quantification of wound gap (C) at 24 h, 48 h, and 72 h after application of scratch wound are shown. Wound gap was expressed as a percentage of initial wound gap. D, E. Migration of A2780 ovarian cancer cells with or without FOXP1 knockdown or overexpression was measured by transwell migration assay. Fluorescence microscope images of the cell migration (bar = 100 μm) (D) and quantification of migrated cells (E) at 12 h are shown.

    Techniques Used: Migration, Over Expression, Wound Healing Assay, Transwell Migration Assay, Fluorescence, Microscopy

    FOXP1 enhances the promoter activity of ABCG2, OCT4, NANOG, and SOX2 A–D. Luciferase reporter assay with ABCG2 promoter (A), OCT4 promoter (B), NANOG promoter (C), or SOX2 promoter (D) with or without FOXP1-binding site deletion (ΔFOXP1) was performed after co-transfection of FOXP1 and each reporter construct into A2780 ovarian cancer cells. E. Western blotting results of A2780 ovarian cancer cells with or without FOXP1 transfection are shown.
    Figure Legend Snippet: FOXP1 enhances the promoter activity of ABCG2, OCT4, NANOG, and SOX2 A–D. Luciferase reporter assay with ABCG2 promoter (A), OCT4 promoter (B), NANOG promoter (C), or SOX2 promoter (D) with or without FOXP1-binding site deletion (ΔFOXP1) was performed after co-transfection of FOXP1 and each reporter construct into A2780 ovarian cancer cells. E. Western blotting results of A2780 ovarian cancer cells with or without FOXP1 transfection are shown.

    Techniques Used: Activity Assay, Luciferase, Reporter Assay, Binding Assay, Cotransfection, Construct, Western Blot, Transfection

    Expression of FOXP1 increases in suspension culture of A2780 ovarian cancer cells A. Bright field images of the spheroid generated from A2780 ovarian cancer cells are shown from day 0 to day 20 of spheroid culture (magnification, ×100, bar = 100 μm). B. Expression of FOXP1 mRNA in the spheroids generated from A2780 ovarian cancer cells is shown by RT-PCR from day 0 to day 20 of spheroid culture. C. Expression of FOXP1 protein in the spheroids generated from A2780 ovarian cancer cells is shown by Western blotting from day 5 to day 20 of spheroid culture. D. Expressions of HIF-1α and FOXP1 in the spheroids generated from A2780 ovarian cancer cells are shown by immunocytochemistry from day 0 to day 20 of spheroid culture (bar = 50 μm). E. Results of Western blotting analysis after treating A2780 spheroid cells with DFO (100 μM) or cobalt chloride (100 μM) are shown. F. Results of quantitative analysis of FOXP1 expression in E. are shown.
    Figure Legend Snippet: Expression of FOXP1 increases in suspension culture of A2780 ovarian cancer cells A. Bright field images of the spheroid generated from A2780 ovarian cancer cells are shown from day 0 to day 20 of spheroid culture (magnification, ×100, bar = 100 μm). B. Expression of FOXP1 mRNA in the spheroids generated from A2780 ovarian cancer cells is shown by RT-PCR from day 0 to day 20 of spheroid culture. C. Expression of FOXP1 protein in the spheroids generated from A2780 ovarian cancer cells is shown by Western blotting from day 5 to day 20 of spheroid culture. D. Expressions of HIF-1α and FOXP1 in the spheroids generated from A2780 ovarian cancer cells are shown by immunocytochemistry from day 0 to day 20 of spheroid culture (bar = 50 μm). E. Results of Western blotting analysis after treating A2780 spheroid cells with DFO (100 μM) or cobalt chloride (100 μM) are shown. F. Results of quantitative analysis of FOXP1 expression in E. are shown.

    Techniques Used: Expressing, Generated, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunocytochemistry

    6) Product Images from "Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair"

    Article Title: Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36506-w

    CRISPR/Cas9 templated gene editing analysis in mouse fibroblasts. ( A ) Overview of the sgRNA + 1 target site with sections of the sense and antisense (AS) ssODN that remove the c.828TAAA insertion and introduce a Kpn I recognition site by a silent C > A substitution. Predicted cut site for Cas9 are indicated by “ ˄ ” and “ ”, with “ ” indicating the strand that is nicked by Cas9D10A. Nucleotide numbers refer to the mutant c.828InsTAAA Fancf gene. ( B ) Overall gene editing efficiencies obtained in mouse fibroblasts depicted as general indel frequencies determined by TIDER (n = 3, one sided Student’s T-test, *p = 0.02). ( C ) Restriction fragment length polymorphism (RFLP) analysis by Kpn I digest of the Fancf locus provides evidence for template-based repair after Cas9, sgRNA + 1 and ssODN transfection (n = 3, one sided Student’s T-test, *p
    Figure Legend Snippet: CRISPR/Cas9 templated gene editing analysis in mouse fibroblasts. ( A ) Overview of the sgRNA + 1 target site with sections of the sense and antisense (AS) ssODN that remove the c.828TAAA insertion and introduce a Kpn I recognition site by a silent C > A substitution. Predicted cut site for Cas9 are indicated by “ ˄ ” and “ ”, with “ ” indicating the strand that is nicked by Cas9D10A. Nucleotide numbers refer to the mutant c.828InsTAAA Fancf gene. ( B ) Overall gene editing efficiencies obtained in mouse fibroblasts depicted as general indel frequencies determined by TIDER (n = 3, one sided Student’s T-test, *p = 0.02). ( C ) Restriction fragment length polymorphism (RFLP) analysis by Kpn I digest of the Fancf locus provides evidence for template-based repair after Cas9, sgRNA + 1 and ssODN transfection (n = 3, one sided Student’s T-test, *p

    Techniques Used: CRISPR, Introduce, Mutagenesis, Transfection

    CRISPR/Cas9 templated gene editing analysis in mouse ES cells. ( A ) Overall gene editing efficiencies obtained in mouse embryonic stem cells depicted as general indel frequencies determined by TIDER (n = 3, one sided Student’s T-test, *p ≤ 0.02). ( B ) TIDE analysis of indel formation at predicted off-target loci after wtCas9 and sgRNA + 1 exposure irrespective of the ssODN orientation (n = 3, one sided Student’s T-test, *p ≤ 0.009). ( C ) Restriction fragment length polymorphism (RFLP) analysis by Kpn I digest of the Fancf locus provides evidence for template-based repair after Cas9 transfection (n = 6, one sided Student’s T-test, *p ≤ 0.02). ( D ) Mouse ES cells show enhanced clonal survival in the presence of 12.5 nM MMC after double-strand break formation by Cas9 and sgRNA + 1 independent of the orientation of the HDR template. A significant difference in relative mESC clone survival was also observed with the antisense (AS) ssODN following Cas9 nickase activity (n = 6, one sided Student’s T-test, *p ≤ 0.025), “M” = mock sgRNA.
    Figure Legend Snippet: CRISPR/Cas9 templated gene editing analysis in mouse ES cells. ( A ) Overall gene editing efficiencies obtained in mouse embryonic stem cells depicted as general indel frequencies determined by TIDER (n = 3, one sided Student’s T-test, *p ≤ 0.02). ( B ) TIDE analysis of indel formation at predicted off-target loci after wtCas9 and sgRNA + 1 exposure irrespective of the ssODN orientation (n = 3, one sided Student’s T-test, *p ≤ 0.009). ( C ) Restriction fragment length polymorphism (RFLP) analysis by Kpn I digest of the Fancf locus provides evidence for template-based repair after Cas9 transfection (n = 6, one sided Student’s T-test, *p ≤ 0.02). ( D ) Mouse ES cells show enhanced clonal survival in the presence of 12.5 nM MMC after double-strand break formation by Cas9 and sgRNA + 1 independent of the orientation of the HDR template. A significant difference in relative mESC clone survival was also observed with the antisense (AS) ssODN following Cas9 nickase activity (n = 6, one sided Student’s T-test, *p ≤ 0.025), “M” = mock sgRNA.

    Techniques Used: CRISPR, Transfection, Activity Assay

    Related Articles

    Amplification:

    Article Title: Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity
    Article Snippet: .. For expression of the Crp protein, the complete P. multocida crp sequence was amplified from P. multocida 0818 chromosomal DNA using the primers pET-crp -F and pET-crp -R. The PCR fragment was then purified and digested with kpn I- HF and BamH I- HF (NEB) and subsequently ligated to the pET-32a expression vector (Novagen Inc., Madison, WI, USA) between the kpn I and BamH I sites to generate pET-32a-crp . .. To complement the crp mutant in P. multocida , the complete crp gene was amplified from P. multocida 0818 genomic DNA using the primers Ccrp -F2/Ccrp -R2, and the amplified fragment was then digested and inserted into the Not I and BamH I sites of a shuttle vector pMC-Express [ ] (kindly donated by Paul R Langford from Imperial College London) to generate pQK176.

    Subcloning:

    Article Title: High-performance chemical- and light-inducible recombinases in mammalian cells and mice
    Article Snippet: .. A similar process was followed as before with the C-terminal CID and LID sub-cloning vectors; however, Kpn I-HF and Eco RI-HF restriction digests (New England Biolabs) were carried out rather than with Mlu I and Bsp EI. .. To swap between CID and LID domains, sequenced fragments were gel isolated from sequenced vectors using Mlu I/Bsp EI or Kpn I-HF/Eco RI-HF and inserted into corresponding CID or LID sub-cloning vectors.

    Construct:

    Article Title: IQGAP1 Negatively Regulates HIV-1 Gag Trafficking and Virion Production
    Article Snippet: .. Flag-tagged Rev-independent HIV-1 Gag domain expression constructs were purchased as gBlocks (IDT; ), digested using KpnI and XhoI (NEB; Cat# R3142 and R0146; respectively) and ligated into pCDNA4/TO, which was digested using the same restriction enzymes. .. PTAPmut plasmid was generated by overlapping PCR using the Gag-flag plasmid and REVi Gag FW together with PTAP2AAA REV primers and REVi Gag REV together with PTAP2AAA FW primers.

    Purification:

    Article Title: Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity
    Article Snippet: .. For expression of the Crp protein, the complete P. multocida crp sequence was amplified from P. multocida 0818 chromosomal DNA using the primers pET-crp -F and pET-crp -R. The PCR fragment was then purified and digested with kpn I- HF and BamH I- HF (NEB) and subsequently ligated to the pET-32a expression vector (Novagen Inc., Madison, WI, USA) between the kpn I and BamH I sites to generate pET-32a-crp . .. To complement the crp mutant in P. multocida , the complete crp gene was amplified from P. multocida 0818 genomic DNA using the primers Ccrp -F2/Ccrp -R2, and the amplified fragment was then digested and inserted into the Not I and BamH I sites of a shuttle vector pMC-Express [ ] (kindly donated by Paul R Langford from Imperial College London) to generate pQK176.

    Polymerase Chain Reaction:

    Article Title: Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair
    Article Snippet: .. In experiments with a template ssODN for homology-directed repair Kpn I-HF (New England Biolabs) digestion reactions were performed on PCR product generated with the same primers. .. In standard RFLP assays, digested PCR products were run on an agarose gel.

    Article Title: The ter Mutation in the Rat Dnd1 Gene Initiates Gonadal Teratomas and Infertility in Both Genders
    Article Snippet: .. The PCR products were digested by adding 0.5 µl Kpn I-HF, 3 µl NEB4 buffer and 0.3 µl BSA (100×) (NEB, Ipswich, MA). .. Cleavage fragments from PCR products of wild type Dnd1 (180 bp and 380 bp) and unchanged 560 bp ter allele products were separated by electrophoresis in a 1.5% agarose gel in 1× TBE buffer.

    Article Title: Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity
    Article Snippet: .. For expression of the Crp protein, the complete P. multocida crp sequence was amplified from P. multocida 0818 chromosomal DNA using the primers pET-crp -F and pET-crp -R. The PCR fragment was then purified and digested with kpn I- HF and BamH I- HF (NEB) and subsequently ligated to the pET-32a expression vector (Novagen Inc., Madison, WI, USA) between the kpn I and BamH I sites to generate pET-32a-crp . .. To complement the crp mutant in P. multocida , the complete crp gene was amplified from P. multocida 0818 genomic DNA using the primers Ccrp -F2/Ccrp -R2, and the amplified fragment was then digested and inserted into the Not I and BamH I sites of a shuttle vector pMC-Express [ ] (kindly donated by Paul R Langford from Imperial College London) to generate pQK176.

    Article Title: Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair
    Article Snippet: .. In experiments with a template ssODN for homology-directed repair Kpn I-HF (New England Biolabs) digestion reactions were performed on PCR product generated with the same primers. .. In standard RFLP assays, digested PCR products were run on an agarose gel.

    Generated:

    Article Title: Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair
    Article Snippet: .. In experiments with a template ssODN for homology-directed repair Kpn I-HF (New England Biolabs) digestion reactions were performed on PCR product generated with the same primers. .. In standard RFLP assays, digested PCR products were run on an agarose gel.

    Article Title: Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair
    Article Snippet: .. In experiments with a template ssODN for homology-directed repair Kpn I-HF (New England Biolabs) digestion reactions were performed on PCR product generated with the same primers. .. In standard RFLP assays, digested PCR products were run on an agarose gel.

    Positron Emission Tomography:

    Article Title: Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity
    Article Snippet: .. For expression of the Crp protein, the complete P. multocida crp sequence was amplified from P. multocida 0818 chromosomal DNA using the primers pET-crp -F and pET-crp -R. The PCR fragment was then purified and digested with kpn I- HF and BamH I- HF (NEB) and subsequently ligated to the pET-32a expression vector (Novagen Inc., Madison, WI, USA) between the kpn I and BamH I sites to generate pET-32a-crp . .. To complement the crp mutant in P. multocida , the complete crp gene was amplified from P. multocida 0818 genomic DNA using the primers Ccrp -F2/Ccrp -R2, and the amplified fragment was then digested and inserted into the Not I and BamH I sites of a shuttle vector pMC-Express [ ] (kindly donated by Paul R Langford from Imperial College London) to generate pQK176.

    Expressing:

    Article Title: IQGAP1 Negatively Regulates HIV-1 Gag Trafficking and Virion Production
    Article Snippet: .. Flag-tagged Rev-independent HIV-1 Gag domain expression constructs were purchased as gBlocks (IDT; ), digested using KpnI and XhoI (NEB; Cat# R3142 and R0146; respectively) and ligated into pCDNA4/TO, which was digested using the same restriction enzymes. .. PTAPmut plasmid was generated by overlapping PCR using the Gag-flag plasmid and REVi Gag FW together with PTAP2AAA REV primers and REVi Gag REV together with PTAP2AAA FW primers.

    Article Title: Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity
    Article Snippet: .. For expression of the Crp protein, the complete P. multocida crp sequence was amplified from P. multocida 0818 chromosomal DNA using the primers pET-crp -F and pET-crp -R. The PCR fragment was then purified and digested with kpn I- HF and BamH I- HF (NEB) and subsequently ligated to the pET-32a expression vector (Novagen Inc., Madison, WI, USA) between the kpn I and BamH I sites to generate pET-32a-crp . .. To complement the crp mutant in P. multocida , the complete crp gene was amplified from P. multocida 0818 genomic DNA using the primers Ccrp -F2/Ccrp -R2, and the amplified fragment was then digested and inserted into the Not I and BamH I sites of a shuttle vector pMC-Express [ ] (kindly donated by Paul R Langford from Imperial College London) to generate pQK176.

    Sequencing:

    Article Title: Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity
    Article Snippet: .. For expression of the Crp protein, the complete P. multocida crp sequence was amplified from P. multocida 0818 chromosomal DNA using the primers pET-crp -F and pET-crp -R. The PCR fragment was then purified and digested with kpn I- HF and BamH I- HF (NEB) and subsequently ligated to the pET-32a expression vector (Novagen Inc., Madison, WI, USA) between the kpn I and BamH I sites to generate pET-32a-crp . .. To complement the crp mutant in P. multocida , the complete crp gene was amplified from P. multocida 0818 genomic DNA using the primers Ccrp -F2/Ccrp -R2, and the amplified fragment was then digested and inserted into the Not I and BamH I sites of a shuttle vector pMC-Express [ ] (kindly donated by Paul R Langford from Imperial College London) to generate pQK176.

    Plasmid Preparation:

    Article Title: Hypoxia-inducible lipid droplet-associated protein inhibits adipose triglyceride lipase
    Article Snippet: .. The EGFP fragment was then excised from the pEGFP-N2 parent vector by enzyme digestion with Kpn I-HF and Not I-HF. .. The vector was gel-purified with QIAquick gel extraction kit (Qiagen) and the fragments of mCherry, sYFP2, or mTurquoise2 ( ) were ligated using T4 DNA ligase (Thermo Fisher Scientific) into the pEGFP-N2 vector, which was digested with Kpn I and Not I.

    Article Title: Identification of the crp gene in avian Pasteurella multocida and evaluation of the effects of crp deletion on its phenotype, virulence and immunogenicity
    Article Snippet: .. For expression of the Crp protein, the complete P. multocida crp sequence was amplified from P. multocida 0818 chromosomal DNA using the primers pET-crp -F and pET-crp -R. The PCR fragment was then purified and digested with kpn I- HF and BamH I- HF (NEB) and subsequently ligated to the pET-32a expression vector (Novagen Inc., Madison, WI, USA) between the kpn I and BamH I sites to generate pET-32a-crp . .. To complement the crp mutant in P. multocida , the complete crp gene was amplified from P. multocida 0818 genomic DNA using the primers Ccrp -F2/Ccrp -R2, and the amplified fragment was then digested and inserted into the Not I and BamH I sites of a shuttle vector pMC-Express [ ] (kindly donated by Paul R Langford from Imperial College London) to generate pQK176.

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    New England Biolabs homology directed repair kpn i hf
    CRISPR/Cas9 templated gene editing analysis in mouse fibroblasts. ( A ) Overview of the sgRNA + 1 target site with sections of the sense and antisense (AS) <t>ssODN</t> that remove the c.828TAAA insertion and introduce a <t>Kpn</t> I recognition site by a silent C > A substitution. Predicted cut site for Cas9 are indicated by “ ˄ ” and “ ”, with “ ” indicating the strand that is nicked by Cas9D10A. Nucleotide numbers refer to the mutant c.828InsTAAA Fancf gene. ( B ) Overall gene editing efficiencies obtained in mouse fibroblasts depicted as general indel frequencies determined by TIDER (n = 3, one sided Student’s T-test, *p = 0.02). ( C ) Restriction fragment length polymorphism (RFLP) analysis by Kpn I digest of the Fancf locus provides evidence for template-based repair after Cas9, sgRNA + 1 and ssODN transfection (n = 3, one sided Student’s T-test, *p
    Homology Directed Repair Kpn I Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CRISPR/Cas9 templated gene editing analysis in mouse fibroblasts. ( A ) Overview of the sgRNA + 1 target site with sections of the sense and antisense (AS) ssODN that remove the c.828TAAA insertion and introduce a Kpn I recognition site by a silent C > A substitution. Predicted cut site for Cas9 are indicated by “ ˄ ” and “ ”, with “ ” indicating the strand that is nicked by Cas9D10A. Nucleotide numbers refer to the mutant c.828InsTAAA Fancf gene. ( B ) Overall gene editing efficiencies obtained in mouse fibroblasts depicted as general indel frequencies determined by TIDER (n = 3, one sided Student’s T-test, *p = 0.02). ( C ) Restriction fragment length polymorphism (RFLP) analysis by Kpn I digest of the Fancf locus provides evidence for template-based repair after Cas9, sgRNA + 1 and ssODN transfection (n = 3, one sided Student’s T-test, *p

    Journal: Scientific Reports

    Article Title: Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair

    doi: 10.1038/s41598-018-36506-w

    Figure Lengend Snippet: CRISPR/Cas9 templated gene editing analysis in mouse fibroblasts. ( A ) Overview of the sgRNA + 1 target site with sections of the sense and antisense (AS) ssODN that remove the c.828TAAA insertion and introduce a Kpn I recognition site by a silent C > A substitution. Predicted cut site for Cas9 are indicated by “ ˄ ” and “ ”, with “ ” indicating the strand that is nicked by Cas9D10A. Nucleotide numbers refer to the mutant c.828InsTAAA Fancf gene. ( B ) Overall gene editing efficiencies obtained in mouse fibroblasts depicted as general indel frequencies determined by TIDER (n = 3, one sided Student’s T-test, *p = 0.02). ( C ) Restriction fragment length polymorphism (RFLP) analysis by Kpn I digest of the Fancf locus provides evidence for template-based repair after Cas9, sgRNA + 1 and ssODN transfection (n = 3, one sided Student’s T-test, *p

    Article Snippet: In experiments with a template ssODN for homology-directed repair Kpn I-HF (New England Biolabs) digestion reactions were performed on PCR product generated with the same primers.

    Techniques: CRISPR, Introduce, Mutagenesis, Transfection

    Genotyping, survival and tumor progression. ( A ) Genotyping. The ter mutation disrupts a Kpn I restriction site used for genotyping of the ter allele performing PCR amplification and restriction digest. Kpn I digest (black arrows) cleaved the PCR product of 560 bp into 380 bp and 180 bp fragments, N negative control. ( B ) Kaplan-Meyer survival analysis of male and female WKY- Dnd1 ter /Ztm rats carrying heterozygous and homozygous ter alleles compared to wild type animals. ( C ) Sizes of TGCTs and OGCTs after 3, 6 and 9 weeks of age in homozygous WKY- Dnd1 ter /Ztm rats.

    Journal: PLoS ONE

    Article Title: The ter Mutation in the Rat Dnd1 Gene Initiates Gonadal Teratomas and Infertility in Both Genders

    doi: 10.1371/journal.pone.0038001

    Figure Lengend Snippet: Genotyping, survival and tumor progression. ( A ) Genotyping. The ter mutation disrupts a Kpn I restriction site used for genotyping of the ter allele performing PCR amplification and restriction digest. Kpn I digest (black arrows) cleaved the PCR product of 560 bp into 380 bp and 180 bp fragments, N negative control. ( B ) Kaplan-Meyer survival analysis of male and female WKY- Dnd1 ter /Ztm rats carrying heterozygous and homozygous ter alleles compared to wild type animals. ( C ) Sizes of TGCTs and OGCTs after 3, 6 and 9 weeks of age in homozygous WKY- Dnd1 ter /Ztm rats.

    Article Snippet: The PCR products were digested by adding 0.5 µl Kpn I-HF, 3 µl NEB4 buffer and 0.3 µl BSA (100×) (NEB, Ipswich, MA).

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

    CRISPR/Cas9 templated gene editing analysis in mouse fibroblasts. ( A ) Overview of the sgRNA + 1 target site with sections of the sense and antisense (AS) ssODN that remove the c.828TAAA insertion and introduce a Kpn I recognition site by a silent C > A substitution. Predicted cut site for Cas9 are indicated by “ ˄ ” and “ ”, with “ ” indicating the strand that is nicked by Cas9D10A. Nucleotide numbers refer to the mutant c.828InsTAAA Fancf gene. ( B ) Overall gene editing efficiencies obtained in mouse fibroblasts depicted as general indel frequencies determined by TIDER (n = 3, one sided Student’s T-test, *p = 0.02). ( C ) Restriction fragment length polymorphism (RFLP) analysis by Kpn I digest of the Fancf locus provides evidence for template-based repair after Cas9, sgRNA + 1 and ssODN transfection (n = 3, one sided Student’s T-test, *p

    Journal: Scientific Reports

    Article Title: Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair

    doi: 10.1038/s41598-018-36506-w

    Figure Lengend Snippet: CRISPR/Cas9 templated gene editing analysis in mouse fibroblasts. ( A ) Overview of the sgRNA + 1 target site with sections of the sense and antisense (AS) ssODN that remove the c.828TAAA insertion and introduce a Kpn I recognition site by a silent C > A substitution. Predicted cut site for Cas9 are indicated by “ ˄ ” and “ ”, with “ ” indicating the strand that is nicked by Cas9D10A. Nucleotide numbers refer to the mutant c.828InsTAAA Fancf gene. ( B ) Overall gene editing efficiencies obtained in mouse fibroblasts depicted as general indel frequencies determined by TIDER (n = 3, one sided Student’s T-test, *p = 0.02). ( C ) Restriction fragment length polymorphism (RFLP) analysis by Kpn I digest of the Fancf locus provides evidence for template-based repair after Cas9, sgRNA + 1 and ssODN transfection (n = 3, one sided Student’s T-test, *p

    Article Snippet: In experiments with a template ssODN for homology-directed repair Kpn I-HF (New England Biolabs) digestion reactions were performed on PCR product generated with the same primers.

    Techniques: CRISPR, Introduce, Mutagenesis, Transfection

    CRISPR/Cas9 templated gene editing analysis in mouse ES cells. ( A ) Overall gene editing efficiencies obtained in mouse embryonic stem cells depicted as general indel frequencies determined by TIDER (n = 3, one sided Student’s T-test, *p ≤ 0.02). ( B ) TIDE analysis of indel formation at predicted off-target loci after wtCas9 and sgRNA + 1 exposure irrespective of the ssODN orientation (n = 3, one sided Student’s T-test, *p ≤ 0.009). ( C ) Restriction fragment length polymorphism (RFLP) analysis by Kpn I digest of the Fancf locus provides evidence for template-based repair after Cas9 transfection (n = 6, one sided Student’s T-test, *p ≤ 0.02). ( D ) Mouse ES cells show enhanced clonal survival in the presence of 12.5 nM MMC after double-strand break formation by Cas9 and sgRNA + 1 independent of the orientation of the HDR template. A significant difference in relative mESC clone survival was also observed with the antisense (AS) ssODN following Cas9 nickase activity (n = 6, one sided Student’s T-test, *p ≤ 0.025), “M” = mock sgRNA.

    Journal: Scientific Reports

    Article Title: Effective CRISPR/Cas9-mediated correction of a Fanconi anemia defect by error-prone end joining or templated repair

    doi: 10.1038/s41598-018-36506-w

    Figure Lengend Snippet: CRISPR/Cas9 templated gene editing analysis in mouse ES cells. ( A ) Overall gene editing efficiencies obtained in mouse embryonic stem cells depicted as general indel frequencies determined by TIDER (n = 3, one sided Student’s T-test, *p ≤ 0.02). ( B ) TIDE analysis of indel formation at predicted off-target loci after wtCas9 and sgRNA + 1 exposure irrespective of the ssODN orientation (n = 3, one sided Student’s T-test, *p ≤ 0.009). ( C ) Restriction fragment length polymorphism (RFLP) analysis by Kpn I digest of the Fancf locus provides evidence for template-based repair after Cas9 transfection (n = 6, one sided Student’s T-test, *p ≤ 0.02). ( D ) Mouse ES cells show enhanced clonal survival in the presence of 12.5 nM MMC after double-strand break formation by Cas9 and sgRNA + 1 independent of the orientation of the HDR template. A significant difference in relative mESC clone survival was also observed with the antisense (AS) ssODN following Cas9 nickase activity (n = 6, one sided Student’s T-test, *p ≤ 0.025), “M” = mock sgRNA.

    Article Snippet: In experiments with a template ssODN for homology-directed repair Kpn I-HF (New England Biolabs) digestion reactions were performed on PCR product generated with the same primers.

    Techniques: CRISPR, Transfection, Activity Assay