Salabi Fatemeh, Nazari Mahmood, Chen Qing, Nimal Jonathan, Tong Jianming, Cao Wen G
J Biotechnol. 2014 Dec 20;192 Pt A:268-80. doi: 10.1016/j.jbiotec.2014.10.038.
Myostatin (MSTN) has previously been shown to negatively regulate the proliferation and differentiation of skeletal muscle cells. Satellite cells are quiescent muscle stem cells that promote muscle growth and repair. Because the mechanism of MSTN in the biology of satellite cells is not well understood, this study was conducted to generate MSTN mono-allelic knockout satellite cells using the zinc-finger nuclease mRNA (MSTN-KO ZFN mRNA) and also to investigate the effect of this disruption on the proliferation and differentiation of sheep primary satellite cells (PSCs). Nineteen biallelic and four mono-allelic knockout cell clones were obtained after sequence analysis. The homologous mono-allelic knockout cells with 5-bp deletion were used to further evaluations. The results demonstrated that mono-allelic knockout of MSTN gene leads to translation inhibition. Real-time quantitative PCR results indicated that knockout of MSTN contributed to an increase in CDK2 and follistatin and a decrease in p21 at the transcript level in proliferation conditions. Moreover, MSTN knockout significantly increased the proliferation of mutant clones (P < 0.01). Consistent with the observed increase in CDK2 and decrease in p21 in cells lacking MSTN, cell cycle analysis showed that MSTN negatively regulated the G1 to S progression. In addition, knockout of myostatin resulted in a remarkable increase in MyoD and MyoG expression under differentiating conditions but had no effect on Myf5 expression. These results expanded our understanding of the regulation mechanism of MSTN. Furthermore, the MSTN-KO ZFN mRNA system in PSCs could be used to generate transgenic sheep in the future.
肌生成抑制素（MSTN）先前已被证明对骨骼肌细胞的增殖和分化具有负调控作用。卫星细胞是促进肌肉生长和修复的静止肌肉干细胞。由于MSTN在卫星细胞生物学中的作用机制尚不清楚，本研究利用锌指核酸酶mRNA（MSTN-KO ZFN mRNA）构建MSTN单等位基因敲除卫星细胞，并研究这种破坏对绵羊原代卫星细胞（PSC）增殖和分化的影响。序列分析后获得了19个双等位基因和4个单等位基因敲除细胞克隆。具有缺失5个碱基对的同源单等位基因敲除细胞用于进一步评估。结果表明，MSTN基因的单等位基因敲除导致翻译抑制。实时定量PCR结果表明，在增殖条件下，敲除MSTN导致转录水平上CDK2和卵泡抑素增加，p21减少。此外，MSTN敲除显著增加了突变克隆的增殖（P < 0.01）。与在缺乏MSTN的细胞中观察到的CDK2增加和p21减少一致，细胞周期分析表明MSTN对G1期到S期的进程具有负调控作用。此外，在分化条件下，敲除肌生成抑制素导致MyoD和MyoG表达显著增加，但对Myf5表达没有影响。这些结果扩展了我们对MSTN调控机制的理解。此外，PSC中的MSTN-KO ZFN mRNA系统将来可用于培育转基因绵羊。
