High-Fidelity Reprogrammed Human IPSCs Have a High Efficacy of DNA Repair and Resemble hESCs in Their MYC Transcriptional Signature

Figure 4

sa-CB-iPSC closely resembled hESC showing greater accuracy of nonhomologous end joining (NHEJ) repair. ((a)(i) and (a)(ii)) Analysis of repair products indicating percentage of misrepair in the in vitro PUC18-based end-joining assay. The misrepair % is calculated by dividing the total # of white colonies by total # of colonies, that is, blue + white, recovered from transformation of the repair products. (a)(i) demonstrates the % misrepair when the dialyzed nuclear lysates from respective cell lines are incubated with PUC18 linearized using EcoR1, giving compatible DNA ends; and (a)(ii) demonstrates the % misrepair when the dialyzed nuclear lysates from respective cell lines are incubated with PUC18 linearized using two restriction endonucleases (Kpn1/SacI), giving noncompatible DNA ends. Statistical significance of the data was determined using one-way ANOVA with Bonferroni posttests to compare all pairs of columns (cell lines). The data is significantly different for H9 or CB6.2 versus iCB9 or iHuF3 (). (b)(i) Shown is a representative gel image of the PCR products from CB6.2 and iCB9 that are redigested with I-Sce1 or left uncut (U). All the S+ products on the gel represent correct repair that restores the I-Sce1 site in the plasmid. (S−) products represent the I-Sce1 resistant repair products, which were cloned into TOP10 competent cells. (b)(ii) The clones, each representing different repair products, were analyzed by sequencing across I-Sce1 junction. Data represents ~10–15 clones analyzed in H9, CB6.2, iCB9, and iHuF3. The data is significantly different for iCB9 versus H9 and CB6.2 (0–5 nt/6–9 nt deletions) or iHuF3 versus H9, CB6.2, and iCB9 (>20 nt deletions) ().

(a)

(b)