肌肉生长抑制素(myostatin, MSTN)是一种骨骼肌生长发育的负调控因子,可以抑制成肌细胞的增殖,是动物品种改良的重要候选基因,该基因突变能够引起骨骼肌的广泛性增生与肥大,产生“双肌”症状,导致动物脂肪分化减少、肌肉含量增加,从而满足市场动物肉品质消费的需求。为了获得“双肌”表型的MSTN基因突变克隆山羊,本研究在山羊MSTN基因第一外显子序列设计并构建TALENs表达载体,转染山羊胎儿成纤维细胞,经嘌呤霉素筛选获得抗性细胞株,通过PCR检测和基因测序筛选MSTN基因突变细胞株作为供核细胞进行山羊体细胞核移植,并鉴定MSTN基因突变类型;通过组织切片分析MSTN基因突变山羊肌肉组织形态结构,监测不同月龄克隆山羊体重并分析其不同生长发育阶段的体重增长趋势。结果表明,共获得109株MSTN基因突变细胞株,突变效率达到79.0% (109/138),其中46株为双等位基因突变,占细胞株总数的33.3% (46/138)。选取4株生长状态较好的MSTN基因突变细胞株(1株为双等位基因纯合突变,3株为非纯合突变)进行体细胞核移植至12只受体山羊,30 d时B超检查出受孕母羊4只,受孕率为33.3% (4/12),妊娠到期分娩2只克隆山羊,测序结果表明M-1克隆山羊MSTN双等位基因中的一个等位基因未产生突变,另一个等位基因缺失3 bp;M-2克隆山羊MSTN双等位基因中的一个等位基因产生1 bp碱基插入,另一个等位基因缺失13 bp,两种突变均造成MSTN在突变位点之后的蛋白序列丢失。此外,M-1克隆山羊肌纤维排列紧密且粗大,每月体重均高于普通野生型山羊,但与普通野生型山羊生长趋势一致,且能够健康发育至成年。本研究利用TALENs技术成功介导山羊MSTN基因定点修饰,并获得MSTN基因突变的山羊胎儿成纤维细胞,将其作为供核细胞成功生产MSTN基因突变克隆山羊,为培育“双肌”表型山羊新品系奠定了技术基础,也为将来其他转基因动物的制备提供了参考方法。

Myostatin (MSTN) is a negative regulator of skeletal muscle growth and development. It can inhibit the proliferation of myoblasts and serve as an important candidate gene for animal breed improvement. Mutations of the MSTN gene can cause extensive skeletal muscle hyperplasia and hypertrophy, resulting in “double muscle” symptoms. This leads to reduction of animal fat differentiation and increase of muscle content, thereby meeting the demand for quality consumption of animal meat in the market. In order to obtain a double-muscle phenotype using mutant MSTN gene in cloned goat, the goat MSTN gene was target-modified by TALENs. In this study, the TALENs expression vector was designed and constructed in the first exon sequence of the goat MSTN gene, which was then transfected into the goat fetal fibroblasts. The resistant cell lines were obtained by puromycin selection, and the cell lines with the MSTN gene mutations were analyzed by PCR and gene sequencing, thereby identifying the mutation type(s). The MSTN gene mutant cell lines were used as the nuclear donor cells in somatic cell nuclear transfer procedures in goats, and The morphological structure of the muscle tissue of the goats with MSTN gene mutations was analyzed by tissue section. The body weight of the cloned goats were monitored at different months of age, which provided the growth trend of their weight at different developmental stages. The results show that a total of 109 MSTN gene mutant cell lines were obtained. The mutation efficiency was 79.0% (109/138), of which 46 were biallelic mutations, accounting for 33.3% (46/138) of the total cell lines. Four MSTN gene mutant cell lines (1 biallelic homozygous mutation, 3 non-homozygous mutations) with good growth status were selected for somatic cell nuclear transfer in 12 recipients, of which 4 were pregnant by B-ultrasound at 30 days, indicating the a 33.3% (4/12) pregnancy rate. Two cloned goats were born at the end of the pregnancy. Sequencing analysis showed that there was no mutation in one allele of the M-1 cloned lamb, and the other allele harbored a 3 bp-deletion. The M-2 cloned lamb harbored a 1 bp base insertion in one allele of the MSTN gene, and a deletion of 13 bp in the other allele, resulting in mutations in both alleles and the loss of the protein-coding sequence of MSTN after the mutation site. In addition, the muscle fibers of cloned M-1 goats are tightly arranged and thick, and their monthly body weight is higher than that of normal wild-type goats. However, it is still consistent with the growth trend of normal wild-type goats and the M-1 goats can develop into healthy adults. In summary, this study showed that goat fetal fibroblasts with the multiple MSTN gene mutations were successfully obtained by TALENs technology, and cloned goats with mutant MSTN genes could be generated by somatic cell nuclear transfer method, thereby providing a technical foundation for the cultivation of the "double muscle" phenotype goats, and serving as a reference method for the preparation of other transgenic animals in the future.