p53 brca1 knockout rpe 1 cells oligonucleotides encoding guide rnas targeting tp53  (Addgene inc)

Journal: NPJ Precision Oncology

Article Title: Examining patient-specific responses to PARP inhibitors in a novel, human induced pluripotent stem cell-based model of breast cancer

doi: 10.1038/s41698-025-00837-5

Figure Lengend Snippet: A Overview for establishing an hiPSC model of breast cancer: primary breast tumor cells are dissociated and reprogrammed into hiPSCs (BC-hiPSCs), followed by differentiation into mammary epithelial cells (BC-hiPSC-MECs) for drug response phenotyping. B Number of BC-hiPSC colonies generated following different reprogramming methodologies, where each row is an independent experiment (OSKM = OCT4 /SOX2/ KLF4 / MYC expression; M87 = breast tumor cell media; B8T/B8 = hiPSC media; n = 2). C Representative phase-contrast images of primary breast cancer cells during reprogramming showing emergence of BC-hiPSC colony. D Flow cytometry assessment of TRA-1-60 and SSEA4 expression in control and BC-hiPSC lines ( n = 3–5). E Flow cytometry assessment of TNNT2 expression in control and BC-hiPSC lines differentiated into cardiomyocytes ( n = 3–6). F Flow cytometry assessment of HNF4A expression in control and BC-hiPSC lines differentiated into hepatocytes ( n = 3–7). G Flow cytometry assessment of NES expression in control and BC-hiPSC lines differentiated into neural progenitor cells ( n = 2-4). H Sanger sequencing of BC-hiPSCs for patient-specific variants in AXIN2 (BC3), BRCA2 (BC6), and BRCA1 (BC8). Variant allele frequency for variants in BC-hiPSCs and isogenic healthy cells from BC6 ( I ) and BC8 ( J ) patients determined by whole genome sequencing. n = experimental replicates, ANOVA, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005, **** P ≤ 0.0001, ns = not significant. Scale bars represent 100 µm.

Article Snippet: Custom, guide-specific CRISPR RNA (crRNA) oligonucleotides targeting BRCA1 were designed using Benchling and CRISPOR design tools (see Supplementary Data for crRNA sequences). crRNA (IDT) and tracrRNA (IDT) were reconstituted in nuclease free duplex buffer (IDT, 11-01-03-01) to a concentration of 200 μM. crRNA and tracrRNA were combined 1:1 (final duplex concentration of 100 μM) and heated at 95 °C for 5 min. After allowing it to cool to RT, the duplex was combined with Alt-R S.p.

Techniques: Generated, Expressing, Flow Cytometry, Control, Sequencing, Variant Assay

Journal: NPJ Precision Oncology

Article Title: Examining patient-specific responses to PARP inhibitors in a novel, human induced pluripotent stem cell-based model of breast cancer

doi: 10.1038/s41698-025-00837-5

Figure Lengend Snippet: A Sanger sequencing of unedited isogenic (ISO) and BRCA1 knockout (KO) hiPSC lines. gRNA target and PAM site indicated. B BRCA1 expression in ISO and BRCA1 KO hiPSC lines by RT-PCR, normalized to ISO ( n = 3). Effect of olaparib treatment (6 days) on ISO and BRCA1 KO hiPSC-MEC viability indicated by resazurin-based kill curve ( C ) and LD 50 ( D ) ( n = 5). Effect of doxorubicin treatment (3 days) on ISO and BRCA1 KO hiPSC-MEC viability indicated by resazurin-based kill curve ( E ) and LD 50 ( F ) ( n = 5). Quantification of γH2AX ( G ) and RAD51 ( H ) staining intensity in ISO and BRCA1 KO hiPSC-MECs treated with indicated concentration of olaparib or DMSO (0 μM), normalized to 0 μM ( n ≥ 74 cells/condition). LD 50 values (mean and SEM) are presented in Supplementary Table . n = experimental replicates, unpaired Student’s T -test, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005, **** P ≤ 0.0001, ns not significant.

Article Snippet: Custom, guide-specific CRISPR RNA (crRNA) oligonucleotides targeting BRCA1 were designed using Benchling and CRISPOR design tools (see Supplementary Data for crRNA sequences). crRNA (IDT) and tracrRNA (IDT) were reconstituted in nuclease free duplex buffer (IDT, 11-01-03-01) to a concentration of 200 μM. crRNA and tracrRNA were combined 1:1 (final duplex concentration of 100 μM) and heated at 95 °C for 5 min. After allowing it to cool to RT, the duplex was combined with Alt-R S.p.

Techniques: Sequencing, Knock-Out, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Concentration Assay

Journal: NPJ Precision Oncology

Article Title: Examining patient-specific responses to PARP inhibitors in a novel, human induced pluripotent stem cell-based model of breast cancer

doi: 10.1038/s41698-025-00837-5

Figure Lengend Snippet: A Sanger sequencing of unedited isogenic control (ISO) hiPSC line and hiPSC line with BRCA1 c.68_69delAG variant introduced (INTRO). gRNA target, PAM site, and silent mutation (*) indicated. B Sanger sequencing of unedited BC8-1 hiPSC line and BC8-1 hiPSC line with BRCA1 c.68_69delAG variant corrected (CORR). gRNA target, PAM site, and silent mutations (*) indicated. Effect of olaparib treatment (6 days) on ISO, INTRO, BC8-1, and CORR hiPSC-MEC viability indicated by resazurin-based kill curve ( C ) and LD 50 ( D ) ( n = 5–6). Effect of doxorubicin treatment (3 days) on ISO, INTRO, BC8-1, and CORR hiPSC-MEC viability indicated by resazurin-based kill curve ( E ) and LD 50 ( F ) ( n = 5-6). Quantification of γH2AX ( G ) and RAD51 ( H ) staining intensity in ISO and INTRO hiPSC-MECs treated with indicated concentration of olaparib or DMSO (0 μM), normalized to 0 μM ( n ≥ 174 cells/condition). Quantification of γH2AX ( I ) and RAD51 ( J ) staining intensity in BC8-1 and CORR hiPSC-MECs treated with indicated concentration of olaparib or DMSO (0 μM), normalized to 0 μM ( n ≥ 140 cells/condition). LD 50 values (mean and SEM) are presented in Supplementary Table . n = experimental replicates, unpaired Student’s T -test, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005, **** P ≤ 0.0001, ns not significant.

Article Snippet: Custom, guide-specific CRISPR RNA (crRNA) oligonucleotides targeting BRCA1 were designed using Benchling and CRISPOR design tools (see Supplementary Data for crRNA sequences). crRNA (IDT) and tracrRNA (IDT) were reconstituted in nuclease free duplex buffer (IDT, 11-01-03-01) to a concentration of 200 μM. crRNA and tracrRNA were combined 1:1 (final duplex concentration of 100 μM) and heated at 95 °C for 5 min. After allowing it to cool to RT, the duplex was combined with Alt-R S.p.

Techniques: Sequencing, Control, Variant Assay, Mutagenesis, Staining, Concentration Assay

Journal: NPJ Precision Oncology

Article Title: Examining patient-specific responses to PARP inhibitors in a novel, human induced pluripotent stem cell-based model of breast cancer

doi: 10.1038/s41698-025-00837-5

Figure Lengend Snippet: Summary of breast cancer patient recruitment and BC-hiPSC generation

Article Snippet: Custom, guide-specific CRISPR RNA (crRNA) oligonucleotides targeting BRCA1 were designed using Benchling and CRISPOR design tools (see Supplementary Data for crRNA sequences). crRNA (IDT) and tracrRNA (IDT) were reconstituted in nuclease free duplex buffer (IDT, 11-01-03-01) to a concentration of 200 μM. crRNA and tracrRNA were combined 1:1 (final duplex concentration of 100 μM) and heated at 95 °C for 5 min. After allowing it to cool to RT, the duplex was combined with Alt-R S.p.

Techniques: