Figure 5

Genetic manipulation in monkey PG-haESCs. (A) Generation of transgenic PG-haESCs via drug selection. After co-transfection of donor PB (Act-RFP) carrying a neomycin-resistant gene (neo^r) and the helper plasmid ACT-PBase into PG-haESCs and antibiotic (G418) selection, almost all neomycin-resistant cells were RFP positive (left). G418-resistant cells were replated and RFP-positive clones were picked for expansion, leading to 12 haploid ESC lines (right). Scale bars, 200 μm. (B) Diagram of PB insertional mutagenesis screening in monkey PG-haESCs. After introducing both PB (PGK-neo) and ACT-PBase into PG-haESCs and G418 selection, survived clones were picked for expansion. Clones with haploid population were selected for further analyses. (C) Four mutated cell lines were established from G418-resistant clones through multiple passages and FACS enrichment for haploid cells. (D) Confirmation of three genes (PRKD1, NIF3L1 and LIPC) mutated in four clones, respectively. Normal PG-haESCs were used as control (lane one). (E) Expression of the NIF3L1 gene measured by qPCR. The expression levels in mutated PG-haESCs were relative to those in ha-ESCs, which were set to 1. ^**P < 0.01.