Production of α1,3-galactosyltransferase targeted pigs using transcription activator-like effector nuclease-mediated genome editing technology

Abstract

Recent developments in genome editing technology using meganucleases demonstrate an efficient method of producing gene edited pigs. In this study, we examined the effectiveness of the transcription activator-like effector nuclease (TALEN) system in generating specific mutations on the pig genome. Specific TALEN was designed to induce a double-strand break on exon 9 of the porcine α1,3-galactosyltransferase (GGTA1) gene as it is the main cause of hyperacute rejection after xenotransplantation. Human decay-accelerating factor (hDAF) gene, which can produce a complement inhibitor to protect cells from complement attack after xenotransplantation, was also integrated into the genome simultaneously. Plasmids coding for the TALEN pair and hDAF gene were transfected into porcine cells by electroporation to disrupt the porcine GGTA1 gene and express hDAF. The transfected cells were then sorted using a biotin-labeled IB4 lectin attached to magnetic beads to obtain GGTA1 deficient cells. As a result, we established GGTA1 knockout (KO) cell lines with biallelic modification (35.0%) and GGTA1 KO cell lines expressing hDAF (13.0%). When these cells were used for somatic cell nuclear transfer, we successfully obtained live GGTA1 KO pigs expressing hDAF. Our results demonstrate that TALEN-mediated genome editing is efficient and can be successfully used to generate gene edited pigs.

Keywords: alpha-1,3-galactosyltransferase; cloned pig; human decay-accelerating factor; knockout; somatic cell nuclear transfer.

Fig. 1. Generation of porcine α1,3-galactosyltransferase (GGTA1) knockout (KO) fibroblasts with transcription activator-like effector nucleases (TALENs). (A) Sequences of the TALEN binding site in the GGTA1 gene. (B) TALEN driven GGTA1 mutations detected by the T7 endonuclease I (T7E1) assay in a cell population isolated using a biotin-labeled IB4 lectin attached to dynabeads magnetic beads. (C) DNA sequencing of TALEN target region in transfected cells. WT, wild type.

Fig. 2. Production of TALEN-mediated piglets by somatic cell cloning. (A) DNA sequencing analysis of GGTA1 TALEN target region in wild-type cells, cloned fetuses and cloned piglets. (B) Genomic PCR of hDAF in cloned piglets. (C) RT-PCR of hDAF in cloned piglets. (D) GGTA1 KO pig expressing hDAF generated from TALEN-mediated donor cells.

Fig. 3. Complement-mediated lysis assay. Monoallelic KO indicates GGTA1 KO heterozygous cells and biallelic KO indicates GGTA1 KO homozygous cells. From number 1 to 6 are cloned piglets derived from hDAF expressing GGTA1 KO cells by nuclear transfer. BSA, bovine serum albumin; NHS, normal human serum.