Highly efficient generation of GGTA1 knockout pigs using a combination of TALEN m RNA and magnetic beads with somatic cell nuclear transfer  

作　　者：FENG Chong LI Xi-rui CUI Hui-ting LONG Chuan LIU Xia TIAN Xing-hua PAN Deng-ke LUO Yu-zhu 

机构地区：[1]Gansu Key Laboratory of Herbivores Animal Biotechnology,College of Animal Science and Technology,Gansu Agricultural University,L anzhou 730070,P.R.China [2]Key Laboratory Farm Animal Genetic Resources and Germplasm Innovation,Ministry of Agriculture/Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing 100193,P.R.China [3]State Key Laboratory of Cellular Stress Biology,School of Life Sciences,Xiamen University,Xiamen 361102,P.R.China [4]Laboratory of Biochemistry and Molecular Biology,College of Life Science,Henan University,Kaifeng 475000,P.R.China

出　　处：《Journal of Integrative Agriculture》2016年第7期1540-1549,共10页农业科学学报（英文版）

基　　金：supported by the National Basic Research Program of China(973 Program)(2015CB554103 and 2011CBA01004)

摘　　要：The transcription activator-like effector nuclease （TALEN） technique combined with the somatic cel nuclear transfer （SCNT） method has been successfuly applied for creating geneticaly modiifed pigs. However, methods for isolating cels with bialelic indels requires further improvement because of the relatively low enrichment efifciency of mutated somatic cels. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modiifed pigs. In this study, an efifcient method to increase the enrichment efifciency of TALEN-mediated bialelic knockout （KO） cels was established, and corresponding geneticaly modiifed 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 α-1,3-galactosyltransferase （GGTA1） gene locus. TALEN mRNA was transfected into the ear ifbroblasts folowed by the enrichment of α-Gal nul cels of minipigs using isolectin B4 （IB4） lectin and magnetic beads. A total of 115 cel colonies were formed and validated to beGGTA1 KO cels by sequencing and 10 bialelic KO cel colonies were used as nuclear donors for SCNT. ThirtyGGTA1 bialelic KO piglets were successfuly delivered and grew normaly. 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-modiifed pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing geneticaly modiifed pig lines with expected genotypes for agricultural breeding and biomedical application.The transcription activator-like effector nuclease （TALEN） technique combined with the somatic cel nuclear transfer （SCNT） method has been successfuly applied for creating geneticaly modiifed pigs. However, methods for isolating cels with bialelic indels requires further improvement because of the relatively low enrichment efifciency of mutated somatic cels. Moreover, little is known regarding the off-target effects of the TALEN system and the heredity of TALEN-modiifed pigs. In this study, an efifcient method to increase the enrichment efifciency of TALEN-mediated bialelic knockout （KO） cels was established, and corresponding geneticaly modiifed 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 α-1,3-galactosyltransferase （GGTA1） gene locus. TALEN mRNA was transfected into the ear ifbroblasts folowed by the enrichment of α-Gal nul cels of minipigs using isolectin B4 （IB4） lectin and magnetic beads. A total of 115 cel colonies were formed and validated to beGGTA1 KO cels by sequencing and 10 bialelic KO cel colonies were used as nuclear donors for SCNT. ThirtyGGTA1 bialelic KO piglets were successfuly delivered and grew normaly. 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-modiifed pigs, 10 mature founders were mated with each other and the mutations were determined to be transmitted to the F1 piglets. We established a robust and safe technology for developing geneticaly modiifed pig lines with expected genotypes for agricultural breeding and biomedical application.

关 键 词：transcription activator-like effector nuclease （TALEN） magnetic beads somatic cel nuclear transfer （SCNT） off-target geneticaly modiifed pigs 

分 类 号：S828[农业科学—畜牧学]