Generation of GGTA1 biallelic knockout pigs via zinc-finger nucleases and somatic cell nuclear transferReport as inadecuate




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Science China Life Sciences

, Volume 57, Issue 2, pp 263–268

First Online: 15 January 2014Received: 12 August 2013Accepted: 11 October 2013DOI: 10.1007-s11427-013-4601-2

Cite this article as: Bao, L., Chen, H., Jong, U. et al. Sci. China Life Sci. 2014 57: 263. doi:10.1007-s11427-013-4601-2

Abstract

Genetically modified pigs are valuable models of human disease and donors of xenotransplanted organs. Conventional gene targeting in pig somatic cells is extremely inefficient. Zinc-finger nuclease ZFN technology has been shown to be a powerful tool for efficiently inducing mutations in the genome. However, ZFN-mediated targeting in pigs has rarely been achieved. Here, we used ZFNs to knock out the porcine α-1, 3-galactosyl-transferase GGTA1 gene, which generates Gal epitopes that trigger hyperacute immune rejection in pig-to-human transplantation. Primary pig fibroblasts were transfected with ZFNs targeting the coding region of GGTA1. Eighteen mono-allelic and four biallelic knockout cell clones were obtained after drug selection with efficiencies of 23.4% and 5.2%, respectively. The biallelic cells were used to produce cloned pigs via somatic cell nuclear transfer SCNT. Three GGTA1 null piglets were born, and one knockout primary fibroblast cell line was established from a cloned fetus. Gal epitopes on GGTA1 null pig cells were completely eliminated from the cell membrane. Functionally, GGTA1 knockout cells were protected from complement-mediated immune attacks when incubated with human serum. This study demonstrated that ZFN is an efficient tool in creating gene-modified pigs. GGTA1 null pigs and GGTA1 null fetal fibroblasts would benefit research and pig-to-human transplantation.

Keywordspig xenotransplantation ZFNs GGTA1 biallelic knockout SCNT Contributed equally to this work

This article is published with open access at link.springer.com

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Author: Lei Bao - HaiDe Chen - UiMyong Jong - CholHo Rim - WenLing Li - XiJuan Lin - Dan Zhang - Qiong Luo - Chun Cui - HeFeng H

Source: https://link.springer.com/







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