Generation of biallelic knock-out sheep via gene-editing and somatic cell nuclear transfer.

Honghui Li,Yubo Qing,Ruoping Zhao,Lei Chen,Guichun Liu,Heng Zhao,Guozhong Zhang,Zhiqiang Hao,Jianxiong Guo,Lili Qing,Wen Wang,Luyao Zeng,Kaixiang Xu,Baoyu Jia,Weirong Pan,Shuanghui Liu,Chaoxiang Lv,Hushan Li,Hongyan Zhao ,Weihua Gui ,Lihua Zhao ,Wood-Hi Cheng ,Hong‐Jiang Wei

doi:10.1038/srep33675

Abstract

Transgenic sheep can be used to achieve genetic improvements in breeds and as an important large-animal model for biomedical research. In this study, we generated a TALEN plasmid specific for ovine MSTN and transfected it into fetal fibroblast cells of STH sheep. MSTN biallelic-KO somatic cells were selected as nuclear donor cells for SCNT. In total, cloned embryos were transferred into 37 recipient gilts, 28 (75.7%) becoming pregnant and 15 delivering, resulting in 23 lambs, 12 of which were alive. Mutations in the lambs were verified via sequencing and T7EI assay, and the gene mutation site was consistent with that in the donor cells. Off-target analysis was performed, and no off-target mutations were detected. MSTN KO affected the mRNA expression of MSTN relative genes. The growth curve for the resulting sheep suggested that MSTN KO caused a remarkable increase in body weight compared with those of wild-type sheep. Histological analyses revealed that MSTN KO resulted in muscle fiber hypertrophy. These findings demonstrate the successful generation of MSTN biallelic-KO STH sheep via gene editing in somatic cells using TALEN technology and SCNT. These MSTN mutant sheep developed and grew normally, and exhibited increased body weight and muscle growth.

Highlights

In breeds that are specialized for the production of superfine or ultrafine wool could be an interesting model for producing more meat in a high quality wool producing animal
transcription activator-like effector nuclease (TALEN) activity was tested, and we found that luciferase activity was increased by 6.2- and 12.6-fold by MSTN-T1 and MSTN-T2, respectively, compared with the activity of the control (Fig. 1B)
Gene editing at the cellular level enables the precise generation of animals with targeted gene modifications while avoiding the mosaicism that accompanies the direct microinjection of fertilized oocytes[17]

Summary

Introduction

In breeds that are specialized for the production of superfine or ultrafine wool could be an interesting model for producing more meat in a high quality wool producing animal. The direct modification of zygotic genomes may have some advantages, including convenient gene manipulation, this strategy may result in mosaic or hypomorphic mutations[16,17,18,19]. In such cases, targeted mutations may not be transmitted to offspring[20], and one or two more rounds of breeding may be required to obtain homozygous animals[16]. We generated genetically modified sheep via gene editing in the somatic cells of STH sheep using TALEN technology followed by SCNT to produce MSTN-knockout (KO) STH sheep. The generation of MSTN-null sheep provides a genetic improvement in sheep breeds for meat production and an important large-animal model for biomedical research on musculoskeletal formation, development, and diseases

Methods

Results

Conclusion