Highly efifcient generation ofGGTA1 knockout pigs using a combination of TALEN mRNA and magnetic beads with somatic cell nuclear transfer

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 α-1,3-galactosyltransferase(GGTA1) gene locus. TALEN m RNA was transfected into the ear fibroblasts followed by the enrichment of α-Gal null cells of minipigs using isolectin B4(IB4) lectin and m agnetic beads. A total of 115 cell c olonies 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 t he fertility and heredity characteristics of TALEN-modified pigs, 10 mature founders w ere 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 genetically modified pig lines with expected genotypes for agricultural breeding and biomedical application.