Additional File 5: Fig. S5. Generation of Rex1-EGFP knock-in porcine PSCs. (A) Targeting diagram showing wild-type (top) and targeted (bottom) pig REX1 alleles generated using the PITCh targeting vector (middle) following CRISPR/Cas9-mediated homology-directed repair as indicated by the dotted lines. The targeting vector consisted of a T2A-EGFP-IRES-PURO-bGHpA cassette (green box) flanked by a 243 bp 5’ homology arm and a 534 bp 3’ homology arm (grey hashed boxes). The homology arms were flanked by inverted CRISPR/Cas9 guide sequences (blue boxes) that matched the endogenous CRIPSR/Cas9 cut site sequence (blue lightning bolts). Following co-electroporation of the targeting vector and Cas9/sgRNA RNP, puro-resistant PSC colonies were generated in which the REX1 stop codon had been replaced with the reporter/selection cassette at the 3’ end of the REX1 coding exon (red box) immediately upstream of the 3’ UTR (brown box). Non-coding genomic sequence and plasmid backbone sequence are represented by thick and thin black lines respectively, and 5’ and 3’ UTRs by brown boxes. Confirmation of correctly targeted clones was performed at both the 5’ and 3’ end of the integration site using forward and reverse primers flanking the 5’ and 3’ homology arms respectively. Expected PCR product sizes are indicated. (B) Five puro-resistant, EGFP+ clones were genotyped by PCR using the primers indicated in panel A. Clones R2, R3 & R4 showed the expected products at both the 5’ and 3’ ends of the integration site. Water and wild-type, parental porcine PSC genomic DNA were used as negative controls (-ve and WT respectively). (C) Compound bright-field and fluorescent image of a REX1-EGFP positive porcine PSC colony. (D) Flow cytometry analysis of porcine REX1-EGFP PSCs and PSCdMs.