文献类型： 外文期刊

作者： Feng Chong ¹ ; Li Xi-rui ² ; Cui Hui-ting ³ ; Long Chuang ² ; Liu Xia ¹ ; Tian Xing-hua; Pan Deng-ke ² ; Luo Yu-zhu ¹ ;

作者机构： 1.Gansu Agr Univ, Coll Anim Sci & Technol, Gansu Key Lab Herbivores Anim Biotechnol, Lanzhou 730070, Peoples R China

2.Chinese Acad Agr Sci, Inst Anim Sci, Minist Agr, Key Lab Farm Anim Genet Resources & Germplasm Inn, Beijing 100193, Peoples R China

3.Xiamen Univ, Sch Life Sci, State Key Lab Cellular Stress Biol, Xiamen 361102, Peoples R China

4.Henan Univ, Coll Life Sci, Lab Biochem & Mol Biol

关键词： transcription activator-like effector nuclease (TALEN);magnetic beads;somatic cell nuclear transfer (SCNT);off-target;genetically modified pigs

期刊名称：JOURNAL OF INTEGRATIVE AGRICULTURE （ 影响因子：2.848； 五年影响因子：2.979 ）

ISSN： 2095-3119

年卷期： 2016 年 15 卷 7 期

页码：

收录情况： SCI

摘要： The transcription activator-like effector nuclease (TALEN) technique combined with the somatic cell nuclear transfer (SCNT) method has been successfully applied for creating genetically modified pigs. However, methods for isolating cells with biallelic indels requires further improvement because of the relatively low enrichment efficiency of mutated somatic cells. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modified pigs. In this study, an efficient method to increase the enrichment efficiency of TALEN-mediated biallelic knockout (KO) cells was established, and corresponding genetically modified pigs with the expected genotype were generated whose off-target effect, fertility and heredity characteristics were aslo evaluated. Two TALEN pairs were constructed to target the porcine alpha-1,3-galactosyltransferase (GGTA1) gene locus. TALEN mRNA was transfected into the ear fibroblasts followed by the enrichment of a-Gal null cells of minipigs using isolectin B4 (IB4) lectin and magnetic beads. A total of 115 cell colonies were formed and validated to be GGTA1 KO cells by sequencing and 10 biallelic KO cell colonies were used as nuclear donors for SCNT. Thirty GGTA1 biallelic KO piglets were successfully delivered and grew normally. Seventeen potential off-target sites were investigated, and no off-target events were detected in the live piglets. To determine the fertility and heredity characteristics of TALEN-modified pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F-1 piglets. We established a robust and safe technology for developing genetically modified pig lines with expected genotypes for agricultural breeding and biomedical application.