Skip to main content
. 2017 Dec;27(12):2061–2071. doi: 10.1101/gr.222521.117

Figure 2.

Figure 2.

Ex vivo single- and multigene knockout in pRosa26-iCas9 fibroblasts. (A) Schematic diagram of ex vivo genome editing experimental workflow. First, pRosa26-iCas9 fibroblasts were isolated from the ear tissues of Cre-dependent Cas9-expressing pigs; second, the isolated pRosa26-iCas9 fibroblasts were infected with lentivirus containing Cre, EGFP, and specific sgRNAs; finally, the genome modifications in infected cells were analyzed at 1 wk posttransduction. (B) Design of sgRNA targeting porcine GGTA1 locus and three representative Sanger sequencing reads of subclones into T-vector from pRosa26-iCas9 fibroblasts. (C) A diagram of lentiviral vectors for Cre recombinase, EGFP, and GGTA1-sgRNA expression. (D) Sanger sequencing of PCR products containing GGTA1-sgRNA targeting site. Upper: pRosa26-iCas9 fibroblasts uninfected with lentivirus; bottom: pRosa26-iCas9 fibroblasts infected with lentivirus containing Cre recombinase, EGFP, and GGTA1-sgRNA. (E) GGTA1-sgRNA-mediated cleavage in wild-type and pRosa26-iCas9 fibroblasts infected or uninfected with lentivirus was analyzed by using a T7EN1 cleavage assay. (F) Western blot analysis for verifying α-Gal epitope and SpCas9 expression in wild-type and pRosa26-iCas9 fibroblasts infected or uninfected with lentivirus. Beta actin was used as a control. (G) Design of sgRNAs targeting early exons of porcine APC, BRCA1, or BRCA2, and three representative Sanger sequencing reads of subclones into T-vector from pRosa26-iCas9 fibroblasts infected with lentivirus AB12. (H) A diagram of lentiviral vector AB12 containing Cre recombinase, EGFP, APC-sgRNA, BRCA1-sgRNA, and BRCA2-sgRNA. (I) Sanger sequencing results of PCR products containing APC-sgRNA, BRCA1-sgRNA, and BRCA2-sgRNA targeting sites.