Highly Efficient Generation of GGTA1 Biallelic Knockout Inbred Mini-Pigs with TALENs

Jige Xin,Huaqiang Yang,Nana Fan,Liangxue Lai,Yi Yang,Zhaoming Liu,Yangzhi Zeng,Bentian Zhao,Xiaoping Li,Zhen Ouyang,Yu Zhao,Quanmei Yan,Qingjian Zou,Jun Song,Xiuhcun (Cindy) Tian

doi:10.1371/journal.pone.0084250

Abstract

Inbred mini-pigs are ideal organ donors for future human xenotransplantations because of their clear genetic background, high homozygosity, and high inbreeding endurance. In this study, we chose fibroblast cells from a highly inbred pig line called Banna mini-pig inbred line (BMI) as donor nuclei for nuclear transfer, combining with transcription activator-like effector nucleases (TALENs) and successfully generated α-1,3-galactosyltransferase (GGTA1) gene biallelic knockout (KO) pigs. To validate the efficiency of TALEN vectors, in vitro-transcribed TALEN mRNAs were microinjected into one-cell stage parthenogenetically activated porcine embryos. The efficiency of indel mutations at the GGTA1-targeting loci was as high as 73.1% (19/26) among the parthenogenetic blastocysts. TALENs were co-transfected into porcine fetal fibroblasts of BMI with a plasmid containing neomycin gene. The targeting efficiency reached 89.5% (187/209) among the survived cell clones after a 10 d selection. More remarkably 27.8% (58/209) of colonies were biallelic KO. Five fibroblast cell lines with biallelic KO were chosen as nuclear donors for somatic cell nuclear transfer (SCNT). Three miniature piglets with biallelic mutations of the GGTA1 gene were achieved. Gal epitopes on the surface of cells from all the three biallelic KO piglets were completely absent. The fibroblasts from the GGTA1 null piglets were more resistant to lysis by pooled complement-preserved normal human serum than those from wild-type pigs. These results indicate that a combination of TALENs technology with SCNT can generate biallelic KO pigs directly with high efficiency. The GGTA1 null piglets with inbred features created in this study can provide a new organ source for xenotransplantation research.

Highlights

Hyperacute rejection (HAR), which is mainly caused by the xenoantigen of galactose-α1,3-galactose (Gal-α1,3Gal), is a major obstacle to pig-to-primate xenotransplantation
The activity was validated by luciferase singlestrand annealing (SSA) recombination assay [20], which showed that transcription activator-like effector nucleases (TALENs) Set#1 had a higher activity over Set#2 (Figure 1C)
To evaluate the targeting effectiveness of TALENs in porcine genome, the mRNAs that were in vitro-transcribed from each pair of TALENs were injected into one-cell stage parthenogenetically activated (PA) porcine embryos, and the genotype of the individual embryos were identified in the blastocyst stage as described in our previous research [13]

Summary

Introduction

Hyperacute rejection (HAR), which is mainly caused by the xenoantigen of galactose-α1,3-galactose (Gal-α1,3Gal), is a major obstacle to pig-to-primate xenotransplantation. Disruption of the α-1,3-galactosyltransferase (GGTA1) gene, which is essential for Gal-α1,3Gal synthesis, is the first step toward overcoming HAR. Most of the KO pigs previously reported were outbred, except those reported by Lai et al [1], whose pig population was difficult to expand because of its low fertility. To address this obstacle, we chose the Banna mini-pig inbred line (BMI) with a high fertility in an effort to create a more applicable pig strain for xenotransplantation research

Methods

Results

Conclusion