One-step generation of multiplex genome editing pigs for xenotransplantation using coselection strategy. (A) Schematic for generating five genes editing xenotransplantation pigs. (Left) The gene-targeting strategy for triple gene knockout (GGTA1, B4GALNT2, and CMAH) and dual gene knock-in (hTHBD and hCD55) in PFFs. The target sites, located in the first exon of GGTA1 and CMAH and the third exon of B4GALNT2, are indicated by arrows. The V2.1G surrogate plasmid is used to select for B4GALNT2 targeting, with selected cells serving as donors for somatic cell nuclear transfer (SCNT) to produce 5Gene xeno-pigs. (B, left) Overview of the DNA sequences at the modification loci in the piglets shown in the right panel. (C) FACS analysis validating the three-gene knockout (3KO) in peripheral blood mononuclear cells (PBMCs). (D) RT-qPCR validation of the two genes knock-in (2KI) in heart and kidney tissues. (E,F) Immunofluorescence (IF) validation of human thrombomodulin (hTBM) and human decay-accelerating factor (hCD55) expression in the kidney (E) and heart (F) of 5Gene pigs.
