Myostatin knockout using zinc-finger nucleases promotes proliferation of ovine primary satellite cells in vitro

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.