FIG. 6.

TW3R human feeders support gene targeting to the AAVS1 locus in human iPSCs. (A) Schematics of AAVS1 locus (lower panel) and the CAG-GFP donor plasmid (upper panel) used for gene targeting. The blue arrow indicates the recognition sites of the ZFN used in this study. After successful HR, endogenous PPP1R12C promoter would drive the expression of Puro through a splice acceptor and a 2A linker (SA-2A-Puro-pA). GFP expression was controlled by the CAG promoter. 5′-probe was used in Southern blot to determine HR occurrence after Sph I (S) digestion of the genomic DNAs. Without HR event, a 6.5 kb fragment between 2 Sph I (S) sites in the WT allele would be hybridized, while a smaller (3.8kb) fragment between the 5′ S site in the genome and the S sites in the donor construct would be observed if HR occurs. As a preliminary screening tool, a pair of PCR primers (indicated by red arrowheads) was designed to amplify a 1 kb fragment if HR occurs. (B) 5×10⁶ BC1 iPSCs were nucleofected with the donor plasmid and the mRNA encoding ZFN genes. After nucleofection, the cells were plated onto either TW3R or DR4 MEF for puromycin selection. Seven days after selection, GFP positive colonies can be observed on both types of feeders. (C) The GFP+ iPSC colonies were more abundant on TW3R feeders than on DR4 MEF feeders. (D) 6 out of 24 puromycin-resistant iPSC clones from TW3R showed a correct PCR band by the primers described in (A), suggesting the occurrence of a HR event in the 6 clones. (E) Southern blot analysis showed that HR occurred in at least one allele of the AAVS1 locus in all 6 candidate clones, as the targeted integration (TI) band of 3.8 kb was observed in all 6 samples. The wild-type (WT) band of 6.5 kb was also observed in clones 10, 12, 16, and 24, indicating that HR occurred in only one allele, while the other allele remained WT. No WT band was observed in clones 5 and 17, suggesting both alleles of the AAVS1 locus were correctly targeted. The band between WT and TI, as observed in clones 12 and 24, suggested the random integration of the donor construct in these clones, in addition to targeted integration. Therefore, clones 10 and 16 are desirable iPSCs with a single allele-corrected targeted at the AAVS1 safe harbor locus. (F) The expanded desirable clones (shown here clone #16) with correct gene targeting maintained undifferentiated morphology and the expression of typical markers associated with pluripotency. (G) H&E staining of teratoma from clone #16 showed tissue types of endoderm (intestinal epithelium), mesoderm (cartilage), and ectoderm (glycogenated squamous epithelium). ZFN, zinc finger nuclease; HR, homologous recombination. Color images available online at www.liebertonline.com/scd