Functional Study Of Different Mutants Of Porcine Myostatin Gene

Myostatin, a transforming growth factor-β family member, is a negative regulator of skeletal muscle development and growth. In this study, we generated four types of transgenic mice overexpressing porcine myostatin missense mutant(pm MS), C313 Y, porcine myostatin that contains mutations at its cleavage site(RSRR), wild-type porcine myostatin propeptide(pp MS) and its mutated form(D75A) to explore their effects on muscle growth in mice. Moreover, the inhibitory activities of porcine myostatin propeptide and its mutated form against murine myostatin was evaluated in vivo by intraperitoneal injection into mice to explore their effects on muscle growth in mice. Besides, we also found that enhanced muscle growth was observed in both pm MS and pp MS transgenic female mice and also in pp MS transgenic male mice. However, there was no enhanced muscle growth observed in pm MS transgenic male mice. To explore why there is such a big difference in muscle growth between pm MS and pp MS transgenic male mice, the expression level of androgen receptor(AR) and its mutant AR45 was measured by Western blot. All the results of this research were as follows:(1) The four types of transgenic mice were confirmed by PCR, RT-PCR, qRT-PCR and Southern Blot. Our data confirm that both types of transgenic mice were successfully generated.(2) The muscle muscle mass weight increased in four types of transgenic mice compared with WT mice except that of the pm MS transgenic male mice. The main skeletal muscle from mice injected with propeptide mutant and mice injected with wild type propeptide was much greater, respectively, than in PBS group.(3) Data indicated the calculation of AR45/AR ratios from density data of AR and AR45 bands showed there is not a significant(p > 0.05) difference between pm MS female mice and wild-type female mice, but there is a significant difference between pm MS male mice and wild-type male mice. These data explain why there is such a big difference in muscle growth between pm MS transgenic male and female mice. Moreover, results indicated that expression of AR and AR45 in pp MS male mice is just the opposite to what was observed in pm MS male mice provides evidence to support our speculation that higher myostatin mature peptide levels in pm MS male mice result in higher levels of AR45, which in turn may reverse the increase in muscle mass induced by mutated myostatin.All in all, these results provide a scientific evidence and basic theory to support future studies to improve the muscle mass in transgenic pigs and to establish a transgenic pig model to be used in biomedical studies for curing muscle disease by genetically manipulating myostatin expression or by regulating myostatin activity.