Fig. 1.

Construction of multiple gene expression vector and identification of GGTA1 (GT) KO/multiple gene KI cell lines. a Schematic of a ZFN-based KI process. The KI vector pLNDT-2A5-Gal has an expression unit for multiple genes with 5′ and 3′ α-gal homology arms at its both end. In the bottom of this figure, the structure of working pLNDT-2A5Gal vector is shown. Primers used for identification of KI cell clones are shown above and below the constructs. b Confirmation of the multiple genes in porcine aortic endothelial cells (PAECs) by RT-PCR. Established the ICAM2-2A5 vector which is the multiple gene expression vector connected by 2A was transfected into PAECs and verified expression of the multiple genes by 2A system before establishment of cell lines. NC: normal PAECs; 2A5: PAECs transfected ICAM2-2A5 vector. c Genotyping of selected cell lines. Targeting vector was transfected into ear fibroblasts from MGH minipigs (mpEF). Four cell lines were established and confirmed by 3 of specific primer sets: 0.8 kb of TG primer sets; 1 kb of 5′ a-Gal primer sets; 1.1 kb of 3′ a-gal primer stets. Among the four cell lines, only two of them were confirmed as transgenic cell lines. 1 normal pig genomic DNA; #2 and 3 transgenic and Knock-in cells; #4 and 5 transgenic cell lines