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Addgene inc doxycycline inducible lentiviral expression vector pcw cas9 puro
Doxycycline Inducible Lentiviral Expression Vector Pcw Cas9 Puro, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 331 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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(A) Family pedigree showing a sibling pair with Fanconi anemia (red circles) who are compound heterozygous for BRCA2 variants c.2330dupA (maternal inheritance) and c.8524C>T (paternal inheritance). Family history of breast cancer (purple, all diagnosed in 60s and 70s), skin cancer (grey), and colon cancer (green, diagnosed at 40 years old). (B) Schematic of BRCA2 domain structure and key interacting proteins (C) Alignment of exon 20 BRCA2 DBD peptide sequence demonstrating it is evolutionary conserved across many species. In green are the aa residues modified by the patient variants, p.W2830_K2833del (c.8488-1G>A) and p.R2842C (c.8524C>T). Purple arrows indicate AA residues that contact DNA . (D) Immunoblot showing BRCA2 levels in WT (RA2985) control, FA-D1 (RA2525), and patient RA3105 and RA3106 LCLs. (E) Quantification of chromosome breaks following DEB treatment of WT (RA2985), FA-A (RA2939), and patient RA3105 and RA3106 LCLs. (F-G) Cell survival assays of patient derived lymphoblast cell lines (LCLs) RA3105, FA-A (RA2939), WT (RA2985), and FA-D1 (RA2525) after MMC and PARP inhibitor olaparib (PARPi) treatment. Relative cell survival was normalized to untreated controls to give percent survival. Error bars indicate s.d. (H) Quantification of chromosome breaks following MMC treatment of BJ wild type fibroblasts, FA-A patient fibroblasts, and HSC62 fibroblasts. (I) Cell survival of HSC62 (c.8488-1G>A) fibroblasts compared to BJ WT fibroblast, FA-A patient fibroblast, complemented FA-A patient cells (RA3087) expressing wild type FANCA (FA-A+A) or empty vector (FA-A+EV). Cells were treated with increasing concentrations of MMC. Relative cell survival was normalized to untreated controls to give the percent survival. Error bars indicate s.d. (J) Cell survival of MMC treated HSC62 uncorrected patient cell line (HSC62 mut ) compared to BJ WT fibroblast and CRISPR/Cas9 corrected wild type HSC62 (HSC62 WT ) clones 1-3. (K-L) Cell survival of BJ WT fibroblasts, and CRISPR/Cas9 targeted BJ fibroblasts: BJ WT fibroblast clone (BRCA2 WT ), c.8488-1G>A BJ clones (BRCA2 8488-1G>A ), c.8524C>T BJ clones (BRCA2 8524C>T) , and exon 20 BRCA2 frameshift mutant (BRCA2 Trun. ). Cells were treated with increasing concentrations of MMC or PARPi. Error bars indicate s.d.

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) Family pedigree showing a sibling pair with Fanconi anemia (red circles) who are compound heterozygous for BRCA2 variants c.2330dupA (maternal inheritance) and c.8524C>T (paternal inheritance). Family history of breast cancer (purple, all diagnosed in 60s and 70s), skin cancer (grey), and colon cancer (green, diagnosed at 40 years old). (B) Schematic of BRCA2 domain structure and key interacting proteins (C) Alignment of exon 20 BRCA2 DBD peptide sequence demonstrating it is evolutionary conserved across many species. In green are the aa residues modified by the patient variants, p.W2830_K2833del (c.8488-1G>A) and p.R2842C (c.8524C>T). Purple arrows indicate AA residues that contact DNA . (D) Immunoblot showing BRCA2 levels in WT (RA2985) control, FA-D1 (RA2525), and patient RA3105 and RA3106 LCLs. (E) Quantification of chromosome breaks following DEB treatment of WT (RA2985), FA-A (RA2939), and patient RA3105 and RA3106 LCLs. (F-G) Cell survival assays of patient derived lymphoblast cell lines (LCLs) RA3105, FA-A (RA2939), WT (RA2985), and FA-D1 (RA2525) after MMC and PARP inhibitor olaparib (PARPi) treatment. Relative cell survival was normalized to untreated controls to give percent survival. Error bars indicate s.d. (H) Quantification of chromosome breaks following MMC treatment of BJ wild type fibroblasts, FA-A patient fibroblasts, and HSC62 fibroblasts. (I) Cell survival of HSC62 (c.8488-1G>A) fibroblasts compared to BJ WT fibroblast, FA-A patient fibroblast, complemented FA-A patient cells (RA3087) expressing wild type FANCA (FA-A+A) or empty vector (FA-A+EV). Cells were treated with increasing concentrations of MMC. Relative cell survival was normalized to untreated controls to give the percent survival. Error bars indicate s.d. (J) Cell survival of MMC treated HSC62 uncorrected patient cell line (HSC62 mut ) compared to BJ WT fibroblast and CRISPR/Cas9 corrected wild type HSC62 (HSC62 WT ) clones 1-3. (K-L) Cell survival of BJ WT fibroblasts, and CRISPR/Cas9 targeted BJ fibroblasts: BJ WT fibroblast clone (BRCA2 WT ), c.8488-1G>A BJ clones (BRCA2 8488-1G>A ), c.8524C>T BJ clones (BRCA2 8524C>T) , and exon 20 BRCA2 frameshift mutant (BRCA2 Trun. ). Cells were treated with increasing concentrations of MMC or PARPi. Error bars indicate s.d.

Article Snippet: To correct the BRCA2 c.8488-1G>A variants in HSC62 fibroblasts, cells were transduced with the pCW-Cas9-Puro (addgene #50661) vector which contains a doxycycline inducible Cas9.

Techniques: Sequencing, Modification, Western Blot, Derivative Assay, Expressing, Plasmid Preparation, CRISPR, Clone Assay, Mutagenesis

(A-B) Chromatograms of Sanger sequencing of DNA derived from a sibling pair and parents confirming the presence of BRCA2 c.2330dupA and c.8524C>T variants identified by whole exome sequencing (WES). (C) BRCA2 structure of the DBD illustrating the location of p.W2830_K2833del and p.R2842C patient variants at the base of the Tower domain and OB2. Structure adapted from Yang et al ., 2005. (D) Immunoblot analysis for FANCI ubiqutination following treatment with 1μM MMC for 24h of. WT (RA2985), FA-A (RA2939), and patient RA3105 and RA3106 LCLs. (E) Metaphase for RA2985 and RA3105 following DEB treatment. (F-G) Cell survival assays of patient derived lymphoblast cell line (LCLs) RA3105, FA-A (RA2939), WT (RA2985), and FA-D1 (RA2525) after (DEB) and camptothecin (CPT) treatment. Survival assays were performed in triplicate. Cells were treated with increasing concentrations of genotoxic agents and counted after 7-10 days in culture. Relative cell survival was normalized to untreated controls to give percent survival. Error bars indicate s.d. (H-N) Cell survival of HSC62 fibroblasts (c.8488-1G>A) to indicated agent compared to BJ WT fibroblast, FA-A patient fibroblast (RA3087), FA-A complemented patient cells expressing wild type FANCA (FA-A+A) or empty vector (FA-A+EV), RAD50 patient fibroblast or FA-D2 (BRCA2/FANCD1) patient fibroblast (FA-D1). Cell survival assays were performed in triplicate. Cells were treated with increasing concentrations of indicated agent. Cell survival was determined by counting cells after 7-9 days in culture. Relative cell survival was normalized to untreated controls to give the percent survival. Error bars indicate s.d. (O) Quantification of chromosome breaks following DEB treatment of BJ wild type fibroblasts, FA-A (FANCA) patient fibroblasts (FA-A mut ), and HSC62 fibroblasts. (P) Chromatograms of PCR amplified gDNA of CRISPR/Cas9 targeted HSC62 fibroblasts. Gene editing reverted the c.8488-1G>A variant either to homozygous WT (HSC62 WT ) or heterozygous WT (HSC62 mut/WT ) at the endogenous locus in HSC62 patient cells. The silent variant that was incorporated to destroy the CRISPR PAM sequence is indicated. (Q)cDNA analysis of HSC62 clones with either homozygous or heterozygous correction of the c.8488-1G>A variant demonstrating rescue of the 12bp deletion of exon 20 that results from alternate splicing. (R) Immunoblot showing BRCA2 levels in CRISPR/CAS9 corrected patient cell line HSC62 WT , uncorrected HSC62 cells (HSC62 mut ), and RA2630 FA-R (RAD51/FANCR) patient fibroblasts. (S-T) Cell survival of HSC62 uncorrected patient cell line (HSC62 mut ) compared to BJ WT fibroblast and CRISPR/Cas9 corrected wild type HSC62 (HSC62 WT ) clone. Error bars indicate s.d.

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A-B) Chromatograms of Sanger sequencing of DNA derived from a sibling pair and parents confirming the presence of BRCA2 c.2330dupA and c.8524C>T variants identified by whole exome sequencing (WES). (C) BRCA2 structure of the DBD illustrating the location of p.W2830_K2833del and p.R2842C patient variants at the base of the Tower domain and OB2. Structure adapted from Yang et al ., 2005. (D) Immunoblot analysis for FANCI ubiqutination following treatment with 1μM MMC for 24h of. WT (RA2985), FA-A (RA2939), and patient RA3105 and RA3106 LCLs. (E) Metaphase for RA2985 and RA3105 following DEB treatment. (F-G) Cell survival assays of patient derived lymphoblast cell line (LCLs) RA3105, FA-A (RA2939), WT (RA2985), and FA-D1 (RA2525) after (DEB) and camptothecin (CPT) treatment. Survival assays were performed in triplicate. Cells were treated with increasing concentrations of genotoxic agents and counted after 7-10 days in culture. Relative cell survival was normalized to untreated controls to give percent survival. Error bars indicate s.d. (H-N) Cell survival of HSC62 fibroblasts (c.8488-1G>A) to indicated agent compared to BJ WT fibroblast, FA-A patient fibroblast (RA3087), FA-A complemented patient cells expressing wild type FANCA (FA-A+A) or empty vector (FA-A+EV), RAD50 patient fibroblast or FA-D2 (BRCA2/FANCD1) patient fibroblast (FA-D1). Cell survival assays were performed in triplicate. Cells were treated with increasing concentrations of indicated agent. Cell survival was determined by counting cells after 7-9 days in culture. Relative cell survival was normalized to untreated controls to give the percent survival. Error bars indicate s.d. (O) Quantification of chromosome breaks following DEB treatment of BJ wild type fibroblasts, FA-A (FANCA) patient fibroblasts (FA-A mut ), and HSC62 fibroblasts. (P) Chromatograms of PCR amplified gDNA of CRISPR/Cas9 targeted HSC62 fibroblasts. Gene editing reverted the c.8488-1G>A variant either to homozygous WT (HSC62 WT ) or heterozygous WT (HSC62 mut/WT ) at the endogenous locus in HSC62 patient cells. The silent variant that was incorporated to destroy the CRISPR PAM sequence is indicated. (Q)cDNA analysis of HSC62 clones with either homozygous or heterozygous correction of the c.8488-1G>A variant demonstrating rescue of the 12bp deletion of exon 20 that results from alternate splicing. (R) Immunoblot showing BRCA2 levels in CRISPR/CAS9 corrected patient cell line HSC62 WT , uncorrected HSC62 cells (HSC62 mut ), and RA2630 FA-R (RAD51/FANCR) patient fibroblasts. (S-T) Cell survival of HSC62 uncorrected patient cell line (HSC62 mut ) compared to BJ WT fibroblast and CRISPR/Cas9 corrected wild type HSC62 (HSC62 WT ) clone. Error bars indicate s.d.

Article Snippet: To correct the BRCA2 c.8488-1G>A variants in HSC62 fibroblasts, cells were transduced with the pCW-Cas9-Puro (addgene #50661) vector which contains a doxycycline inducible Cas9.

Techniques: Sequencing, Derivative Assay, Western Blot, Expressing, Plasmid Preparation, Amplification, CRISPR, Variant Assay, Clone Assay

(A) cDNA sequencing of BRCA2 DBD CRISPR/Cas9 targeted BJ fibroblasts demonstrating that homozygous c.8488-1G>A variant in BJ fibroblast clones result in the usage of an alternative splice site donor and a 12bp deletion at the start of BRCA2 exon 20 (as seen in patient derived HSC62 fibroblasts). (B) cDNA sequencing of BRCA2 DBD CRISPR/Cas9 targeted BJ fibroblasts demonstrating the c.8524C>T missense variant and silent mutation introduced by CRISPR/Cas9 targeting. (C) Immunoblot showing BRCA2 levels in bulk BJ WT fibroblasts, BRCA2 WT fibroblast clone, c.8488-1G>A BJ clones 1-3, c.8524C>T BJ clones 1-2, and BRCA2 Trun. clones 1-2. (D-E) Cell survival of BJ WT fibroblasts, BJ WT fibroblast clone (BRCA2 WT ), c.8488-1G>A BJ clones, c.8524C>T BJ clones, and exon 20 BRCA2 frameshift mutant (BRCA2 Trun. ). Cell survival assays were performed in triplicate. Cells were treated with increasing concentrations of CPT or aphidicolin. Cell survival was determined by counting cells after 7-9 days in culture. Relative cell survival was normalized to untreated controls to give the percent survival. Error bars indicate s.d. (F-G) Representative images of RAD51 foci in HSC62 uncorrected patient cell line (HSC62 mut ) compared to BJ WT fibroblast and CRISPR/Cas9 corrected wild type HSC62 (HSC62 mut/WT or HSC62 WT ) clones, 8h post IR and 24h post MMC. Detected by immunofluorescence with anti-RAD51antibody. Quantification in and . (H) Quantification of RAD51 foci in isogenic BJ fibroblasts clones 8h, 24h and 48h following 1h treatment with 3 μM MMC of BJ WT fibroblasts, BJ WT fibroblast clone (BRCA2 WT ), c.8488-1G>A BJ clones 2-3, c.8524C>T BJ clones 1-2, and a BRCA2 truncation mutant, c.8531dupA ( BRCA2 Trun ). Error bars indicate s.d. of three independent experiments (≥200 cells per experiment). (I) Representative images of RAD51 foci in isogenic BJ fibroblasts clones, 24h post 1h treatment with 3 μM MMC, detected by immunofluorescence with anti-RAD51antibody. Third row images are individual cells enlarged to better demonstrate differences in RAD51 foci size. Quantified in .

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) cDNA sequencing of BRCA2 DBD CRISPR/Cas9 targeted BJ fibroblasts demonstrating that homozygous c.8488-1G>A variant in BJ fibroblast clones result in the usage of an alternative splice site donor and a 12bp deletion at the start of BRCA2 exon 20 (as seen in patient derived HSC62 fibroblasts). (B) cDNA sequencing of BRCA2 DBD CRISPR/Cas9 targeted BJ fibroblasts demonstrating the c.8524C>T missense variant and silent mutation introduced by CRISPR/Cas9 targeting. (C) Immunoblot showing BRCA2 levels in bulk BJ WT fibroblasts, BRCA2 WT fibroblast clone, c.8488-1G>A BJ clones 1-3, c.8524C>T BJ clones 1-2, and BRCA2 Trun. clones 1-2. (D-E) Cell survival of BJ WT fibroblasts, BJ WT fibroblast clone (BRCA2 WT ), c.8488-1G>A BJ clones, c.8524C>T BJ clones, and exon 20 BRCA2 frameshift mutant (BRCA2 Trun. ). Cell survival assays were performed in triplicate. Cells were treated with increasing concentrations of CPT or aphidicolin. Cell survival was determined by counting cells after 7-9 days in culture. Relative cell survival was normalized to untreated controls to give the percent survival. Error bars indicate s.d. (F-G) Representative images of RAD51 foci in HSC62 uncorrected patient cell line (HSC62 mut ) compared to BJ WT fibroblast and CRISPR/Cas9 corrected wild type HSC62 (HSC62 mut/WT or HSC62 WT ) clones, 8h post IR and 24h post MMC. Detected by immunofluorescence with anti-RAD51antibody. Quantification in and . (H) Quantification of RAD51 foci in isogenic BJ fibroblasts clones 8h, 24h and 48h following 1h treatment with 3 μM MMC of BJ WT fibroblasts, BJ WT fibroblast clone (BRCA2 WT ), c.8488-1G>A BJ clones 2-3, c.8524C>T BJ clones 1-2, and a BRCA2 truncation mutant, c.8531dupA ( BRCA2 Trun ). Error bars indicate s.d. of three independent experiments (≥200 cells per experiment). (I) Representative images of RAD51 foci in isogenic BJ fibroblasts clones, 24h post 1h treatment with 3 μM MMC, detected by immunofluorescence with anti-RAD51antibody. Third row images are individual cells enlarged to better demonstrate differences in RAD51 foci size. Quantified in .

Article Snippet: To correct the BRCA2 c.8488-1G>A variants in HSC62 fibroblasts, cells were transduced with the pCW-Cas9-Puro (addgene #50661) vector which contains a doxycycline inducible Cas9.

Techniques: Sequencing, CRISPR, Variant Assay, Clone Assay, Derivative Assay, Mutagenesis, Western Blot, Immunofluorescence

(A) Immunofluorescence images of RAD51 foci, 8h following 12 Gy ionizing radiation (IR) of BJ WT fibroblast and patient derived HSC62 fibroblast, detected with anti-RAD51 antibody. Third row images are individual cells enlarged to better demonstrate differences in RAD51 foci size. (B) Quantification of RAD51 foci 1h, 8h, and 24h following 12 Gy IR of BJ WT fibroblast and HSC62 fibroblast. Error bars indicate s.d. of two independent experiments (≥200 cells per experiment). (C) Quantification of RAD51 foci 8h after 12 Gy IR of BJ WT fibroblast, wild type HSC62 - (HSC62 WT ) clones 1-3, and HSC62 uncorrected patient cell line (HSC62 mut ). (D) Quantification of RAD51 foci 24h following 1h treatment with 3 µM MMC. Error bars indicate s.d. of three independent experiments (≥200 cells per experiment). (E) Quantification of RAD51 foci in isogenic BJ fibroblasts clones at 1h, 8h and 24h following 6 Gy IR of BJ WT fibroblasts, BJ WT fibroblast clone (BRCA2 WT ), BRCA2 8488-1G>A BJ clones 2-3, BRCA2 8524C>T BJ clones 1-2, and a BRCA2 homozygous truncation mutant, c.8531dupA (BRCA2 Trun ). Error bars indicate s.d. of three independent experiments (≥200 cells per experiment) (F) Representative images of RAD51 foci in isogenic BJ fibroblasts clones, 8h post 6 Gy IR, detected by immunofluorescence with anti-RAD51 antibody. Third row images are individual cells enlarged to better demonstrate differences in RAD51 foci size. (G) Quantification of RPA foci 24h following 1h treatment with 3 μM MMC of BJ WT fibroblast, CRISPR/Cas9 corrected wild type HSC62 clones (HSC62 WT ), and HSC62 uncorrected patient cell line (HSC62 mut ). (H) Quantification of RPA foci 24h following 1h treatment with 3 μM MMC in HSC62 mut cells depleted of DNA2, MRE11, EXO1, CTIP, WRN, or BLM by siRNA compared to luciferase control (Luc). Error bars indicate s.d. of four independent experiments. (I) Immunoblot analysis of RPA phosphorylation in isogenic BJ fibroblasts clones 24h post 1h treatment with 3 μM MMC. BRCA2 WT , BRCA2 8524C>T , and BRCA2 8488-1G>A BJ fibroblast cells were transfected with siRNA control luciferase (Luc) or siRNAs targeting DNA2 or WRN. (J-K) MMC cell survival of BJ BRCA2 WT , BRCA2 8488-1G>A , and BRCA2 8524C>T fibroblasts overexpressing (OE) WT RAD51 or empty vector (EV) control. Relative cell survival was normalized to untreated controls to give percent survival. Error bars indicate s.d.

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) Immunofluorescence images of RAD51 foci, 8h following 12 Gy ionizing radiation (IR) of BJ WT fibroblast and patient derived HSC62 fibroblast, detected with anti-RAD51 antibody. Third row images are individual cells enlarged to better demonstrate differences in RAD51 foci size. (B) Quantification of RAD51 foci 1h, 8h, and 24h following 12 Gy IR of BJ WT fibroblast and HSC62 fibroblast. Error bars indicate s.d. of two independent experiments (≥200 cells per experiment). (C) Quantification of RAD51 foci 8h after 12 Gy IR of BJ WT fibroblast, wild type HSC62 - (HSC62 WT ) clones 1-3, and HSC62 uncorrected patient cell line (HSC62 mut ). (D) Quantification of RAD51 foci 24h following 1h treatment with 3 µM MMC. Error bars indicate s.d. of three independent experiments (≥200 cells per experiment). (E) Quantification of RAD51 foci in isogenic BJ fibroblasts clones at 1h, 8h and 24h following 6 Gy IR of BJ WT fibroblasts, BJ WT fibroblast clone (BRCA2 WT ), BRCA2 8488-1G>A BJ clones 2-3, BRCA2 8524C>T BJ clones 1-2, and a BRCA2 homozygous truncation mutant, c.8531dupA (BRCA2 Trun ). Error bars indicate s.d. of three independent experiments (≥200 cells per experiment) (F) Representative images of RAD51 foci in isogenic BJ fibroblasts clones, 8h post 6 Gy IR, detected by immunofluorescence with anti-RAD51 antibody. Third row images are individual cells enlarged to better demonstrate differences in RAD51 foci size. (G) Quantification of RPA foci 24h following 1h treatment with 3 μM MMC of BJ WT fibroblast, CRISPR/Cas9 corrected wild type HSC62 clones (HSC62 WT ), and HSC62 uncorrected patient cell line (HSC62 mut ). (H) Quantification of RPA foci 24h following 1h treatment with 3 μM MMC in HSC62 mut cells depleted of DNA2, MRE11, EXO1, CTIP, WRN, or BLM by siRNA compared to luciferase control (Luc). Error bars indicate s.d. of four independent experiments. (I) Immunoblot analysis of RPA phosphorylation in isogenic BJ fibroblasts clones 24h post 1h treatment with 3 μM MMC. BRCA2 WT , BRCA2 8524C>T , and BRCA2 8488-1G>A BJ fibroblast cells were transfected with siRNA control luciferase (Luc) or siRNAs targeting DNA2 or WRN. (J-K) MMC cell survival of BJ BRCA2 WT , BRCA2 8488-1G>A , and BRCA2 8524C>T fibroblasts overexpressing (OE) WT RAD51 or empty vector (EV) control. Relative cell survival was normalized to untreated controls to give percent survival. Error bars indicate s.d.

Article Snippet: To correct the BRCA2 c.8488-1G>A variants in HSC62 fibroblasts, cells were transduced with the pCW-Cas9-Puro (addgene #50661) vector which contains a doxycycline inducible Cas9.

Techniques: Immunofluorescence, Derivative Assay, Clone Assay, Mutagenesis, CRISPR, Luciferase, Western Blot, Transfection, Plasmid Preparation

(A) Images of RPA foci in HSC62 uncorrected patient cell line (HSC62 mut ) compared to BJ WT fibroblast and CRISPR/Cas9 corrected wild type HSC62 (HSC62 mut/WT or HSC62 WT ) clones, 24h post 1h treatment with 3 μM MMC. Detected by immunofluorescence with anti-RPA32 antibody. Quantification in . (B)Immunoblot analysis of MRE11 and CTIP siRNA depletion for cells utilized in . (C) qRT-PCR of DNA2, EXO1, WRN, and BLM expression levels of cells in . Error bars are s.d. (D) Quantification of RPA foci in isogenic BJ fibroblasts clones 8h, 24h and 48h following 1h treatment with 3 μM MMC of BJ WT fibroblasts, BJ WT fibroblast (BRCA2 WT ), c.8488-1G>A BJ clones 2-3, c.8524C>T BJ clones 1-2, and a BJ BRCA2 truncation mutant (BRCA2 Trun. ). Error bars indicate s.d. of three independent experiments (≥200 cells per experiment). (E) Representative images of RPA foci in isogenic BJ fibroblasts clones, 24h post 1h treatment with 3 uM MMC, detected by immunofluorescence with anti-RPA32 antibody. Third row images are individual cells enlarged to better demonstrate RPA foci. Quantified in . (F) Quantification of RPA foci in isogenic BJ fibroblasts clones 24h following 1h treatment with 3 μM MMC in cells depleted of DNA2 or WRN by siRNA. Error bars indicate s.d of two independent experiments. (G-H) qRT-PCR of DNA2 and WRN expression levels of cells utilized in and . Error bars indicate s.d. (I) Immunoblot analysis of WT RAD51 overexpression in BRCA2 8524C>T and BRCA2 8488-1G>A BJ fibroblast cells used in and . (J) Quantification of RPA foci 24h following 1h treatment with 3 μM MMC in cells expressing WT RAD51 or EV control. Representative data of 3 independent experiments. (K) Quantification of RPA foci in isogenic BJ fibroblasts clones 24h following 1h treatment with 3 uM MMC in cells depleted of SLX4 or MUS81 by siRNA. Error bars indicate s.d of two independent experiments. (L) Immunoblot analysis of MUS81 depletion for cells utilized in . (M) qRT-PCR of SLX4 expression levels of cells utilized in . Error bars indicate s.d. (N-O) qRT-PCR of DNA2 and WRN expression levels of FA-A fibroblast cell lines utilized in . Error bars indicate s.d. (P-Q) qRT-PCR of DNA2 and BLM expression levels of FA-A fibroblast cell lines utilized in . Error bars indicate s.d.

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) Images of RPA foci in HSC62 uncorrected patient cell line (HSC62 mut ) compared to BJ WT fibroblast and CRISPR/Cas9 corrected wild type HSC62 (HSC62 mut/WT or HSC62 WT ) clones, 24h post 1h treatment with 3 μM MMC. Detected by immunofluorescence with anti-RPA32 antibody. Quantification in . (B)Immunoblot analysis of MRE11 and CTIP siRNA depletion for cells utilized in . (C) qRT-PCR of DNA2, EXO1, WRN, and BLM expression levels of cells in . Error bars are s.d. (D) Quantification of RPA foci in isogenic BJ fibroblasts clones 8h, 24h and 48h following 1h treatment with 3 μM MMC of BJ WT fibroblasts, BJ WT fibroblast (BRCA2 WT ), c.8488-1G>A BJ clones 2-3, c.8524C>T BJ clones 1-2, and a BJ BRCA2 truncation mutant (BRCA2 Trun. ). Error bars indicate s.d. of three independent experiments (≥200 cells per experiment). (E) Representative images of RPA foci in isogenic BJ fibroblasts clones, 24h post 1h treatment with 3 uM MMC, detected by immunofluorescence with anti-RPA32 antibody. Third row images are individual cells enlarged to better demonstrate RPA foci. Quantified in . (F) Quantification of RPA foci in isogenic BJ fibroblasts clones 24h following 1h treatment with 3 μM MMC in cells depleted of DNA2 or WRN by siRNA. Error bars indicate s.d of two independent experiments. (G-H) qRT-PCR of DNA2 and WRN expression levels of cells utilized in and . Error bars indicate s.d. (I) Immunoblot analysis of WT RAD51 overexpression in BRCA2 8524C>T and BRCA2 8488-1G>A BJ fibroblast cells used in and . (J) Quantification of RPA foci 24h following 1h treatment with 3 μM MMC in cells expressing WT RAD51 or EV control. Representative data of 3 independent experiments. (K) Quantification of RPA foci in isogenic BJ fibroblasts clones 24h following 1h treatment with 3 uM MMC in cells depleted of SLX4 or MUS81 by siRNA. Error bars indicate s.d of two independent experiments. (L) Immunoblot analysis of MUS81 depletion for cells utilized in . (M) qRT-PCR of SLX4 expression levels of cells utilized in . Error bars indicate s.d. (N-O) qRT-PCR of DNA2 and WRN expression levels of FA-A fibroblast cell lines utilized in . Error bars indicate s.d. (P-Q) qRT-PCR of DNA2 and BLM expression levels of FA-A fibroblast cell lines utilized in . Error bars indicate s.d.

Article Snippet: To correct the BRCA2 c.8488-1G>A variants in HSC62 fibroblasts, cells were transduced with the pCW-Cas9-Puro (addgene #50661) vector which contains a doxycycline inducible Cas9.

Techniques: CRISPR, Clone Assay, Immunofluorescence, Western Blot, Quantitative RT-PCR, Expressing, Mutagenesis, Over Expression

(A) Chromatograms of BRCA2 CRISPR/Cas9 generated HEK293T clones aligned to WT. A frameshift Exon 20 BRCA2 mutant (BRCA2 Trun. ) was generated by homozygous single base pair insertion as a result of CRISPR/Cas9 targeting. BRCA2 exon 20 variants, c.8524C>T and c.8488-1G>A, and Exon 27 p.S3291A (c.9871T>G) clones were generated by targeting the respective BRCA2 exon with CRISPR/Cas9 and a 100bp ssDNA template donor. Where applicable, silent mutations are indicated. Cell lines utilized in . (B) Immunoblot showing BRCA2 levels in WT HEK293T cells and BRCA2 mutant HEK293T clones: c.8531dupA ( BRCA2 Trun ), c.8524C>T (clones 1-2), c.8488-1G>A (clones 1-3), and p.S3291A (clones 1-3). Cells utilized in . (C) qRT-PCR of BRCA2 expression levels of cells utilized in . Error bars are s.d. (D) Immunoblot of RAD51 knockdown for HEK293T cells used in . (E) SCE assay in BJ WT fibroblast and HSC62 fibroblast following depletion of BLM. (F) Representative images of SCEs in BJ WT fibroblast and HSC62 fibroblast metaphases. (G) qRT-PCR of BLM expression levels in cells described in E. Error bars indicate s.d. (H) Sister chromatid exchange (SCE) assay in BJ BRCA2 WT and BRCA2 Trun fibroblasts following treatment with MMC (0.05 μg/ml or 0.1 μg/ml). (I) Immunoblot analysis of MRE11 depletion for cells utilized in ,F. (J) qRT-PCR of DNA2 expression levels of cells utilized in ,F. Error bars indicate s.d.

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet: (A) Chromatograms of BRCA2 CRISPR/Cas9 generated HEK293T clones aligned to WT. A frameshift Exon 20 BRCA2 mutant (BRCA2 Trun. ) was generated by homozygous single base pair insertion as a result of CRISPR/Cas9 targeting. BRCA2 exon 20 variants, c.8524C>T and c.8488-1G>A, and Exon 27 p.S3291A (c.9871T>G) clones were generated by targeting the respective BRCA2 exon with CRISPR/Cas9 and a 100bp ssDNA template donor. Where applicable, silent mutations are indicated. Cell lines utilized in . (B) Immunoblot showing BRCA2 levels in WT HEK293T cells and BRCA2 mutant HEK293T clones: c.8531dupA ( BRCA2 Trun ), c.8524C>T (clones 1-2), c.8488-1G>A (clones 1-3), and p.S3291A (clones 1-3). Cells utilized in . (C) qRT-PCR of BRCA2 expression levels of cells utilized in . Error bars are s.d. (D) Immunoblot of RAD51 knockdown for HEK293T cells used in . (E) SCE assay in BJ WT fibroblast and HSC62 fibroblast following depletion of BLM. (F) Representative images of SCEs in BJ WT fibroblast and HSC62 fibroblast metaphases. (G) qRT-PCR of BLM expression levels in cells described in E. Error bars indicate s.d. (H) Sister chromatid exchange (SCE) assay in BJ BRCA2 WT and BRCA2 Trun fibroblasts following treatment with MMC (0.05 μg/ml or 0.1 μg/ml). (I) Immunoblot analysis of MRE11 depletion for cells utilized in ,F. (J) qRT-PCR of DNA2 expression levels of cells utilized in ,F. Error bars indicate s.d.

Article Snippet: To correct the BRCA2 c.8488-1G>A variants in HSC62 fibroblasts, cells were transduced with the pCW-Cas9-Puro (addgene #50661) vector which contains a doxycycline inducible Cas9.

Techniques: CRISPR, Generated, Clone Assay, Mutagenesis, Western Blot, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Distinct roles of BRCA2 in replication fork protection in response to hydroxyurea and DNA interstrand crosslinks

doi: 10.1101/811968

Figure Lengend Snippet:

Article Snippet: To correct the BRCA2 c.8488-1G>A variants in HSC62 fibroblasts, cells were transduced with the pCW-Cas9-Puro (addgene #50661) vector which contains a doxycycline inducible Cas9.

Techniques: CRISPR

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