Generation And Investigation Of Myostatin Function Deficient Mouse Models

Myostatin is a negative regulator of skeletal muscle growth and inactivation of myostatin can increase growth and meat production of animals, thus myostatin gene is regarded as an important candidate gene for livestock breeding by genetic engineering. Previous studies have shown that there are two ways of inhibiting myostatin function to produce muscle hypertrophy phenotype:one is transgenic overexpression of proteins binding to myostatin as an inhibitor, such as myostatin propeptide; another way is knockout of myostatin gene by gene targeting. Sheep is an important livestock species and have economic values in meat and wool production, thus improving the trait of meat production by genetic modification may be significant for breeding new varieties of sheep. The objective of this study was to investigate the effects of overexpression of ovine myostatin propeptide and knockout of myostatin gene on muscle growth phenotype in mouse models, with the purpose of providing basis for future genetic engineering in sheep. Main results of the study are shown as following:1. Overexpreesion of ovine myostatin propeptide in transgenic micecDNA of ovine myostatin propeptide was cloned and a site mutation was introduced into the sequence for improving the inhibiting effect.In vitro A204cells assay showed that overexpression of ovine myostatin propeptide could block endogenous myostatin function. In vivo injection of ovine myostatin propeptide overexpression plasmid could cause weight gain and myofiber hypertrophy in mice. For controllable expression of ovine myostatin propeptide, a plasmid for tetracycline-inducible muscle specific expression was constructed and transgenic mice was produced with this plasmid. Tetracyline-inducible expression was achieved in both cells in vitro and in transgenic mice in vivo, and tight inducibility and tissue-specificity of the expression were proved. Phenotype analysis showed that treatment of tetracycline had no significant effects on body weight and muscle mass in transgenic mice, but did induce myofiber hypertrophy. These results indicated that conditional inhibition of myostatin function could be achieved by controllable overexpression of myostatin propeptide in mammals and this strategy may be applicable for genetic engineering in sheep.2. Knockout (KO) of myostatin in miceThe recently developed CRISPR/Cas9system was used to knockout myostatin gene in mice. At the same time, fibroblast growth factor5(fgf5) gene, which is a regulator of hair growth, was also knocked out in this study. sgRNAs were designed for targeting DNA sequences in highly conserved regions of both genes and used to produce genetically modified mice by pronuclear microinjection. The results showed that20%of the offspring bore double-gene mutations and no significant off-target effect was detected in their genomes. Phenotype analysis showed that muscle overgrowth was observed in myostatin KO mice and abnormal long hair was seen in fgf5KO mice. Both muscle and hair overgrowth phenotypes appeared simultaneously in myostatin/fgf5double KO mice, however myostatin/fef5double KO mice showed lower body size and viability. In subsequent analysis, an interaction between myostatin and fgf5might play an influence on growth or energy metabolism in postnatal double KO mouse. Taken together, the results demonstrated that one-step knockout of both myostatin and fgf5gene in mammals using CRISPR/Cas9system was efficient and feasible, thus providing guidance for future gene knockout in other species, like sheep.In summary, this study has produced two types of mouse models and explored the potential of transgenic overexpression of ovine myostatin propeptide and knockout of myostatin/fgf5gene in improving the related phenotypes, such as muscle growth. The results provided technical and theoretical basis for future livestock breeding by using these genetic engineering technologies.