Affection Of Myostatin Gene On Chicken Skeletal Muscle Development Studied By Transgenic Technology

Myostatin (GDF-8) is a member of transforming growth factor beta (TGF-β) superfamily, which negatively regulates skeletal muscle growth and development. Many new functions of myostatin have been gradually found. Myostatin could play important roles by regulating the skeletal myogenesis, development, regeneration, atrophy, and degradation. For a better understanding of molecular mechanism of myostatin function in embryogenesis and skeletal muscle development in vivo, chicken is selected as the study object in this experiment. Primary studies on chicken myostatin were carried out in vivo and in vitro, respectively. The experiment processes are as follows:We construct a technology platform of transgenic chicken by using retrovirus. Marker gene (enhanced green fluorescent protein(EGFP)) was cloned into retroviral expression vectors. Vesicular stomatitis virus G glycoprotein(VSV-G) pseudotyped GFP-retrovirus were packaged in phoenix or HEK293T cells. Chicken embryonic myoblasts (CEM) were infected for evaluating the infection efficiency, integration and transcription activity of packaged retrovirus.Then large-scale production of retrovirus were packaged and concentrated, and new hatching eggs were injected with the concentrated retrovirus and hatched. Genomic DNA of 5 days chicken embryos and different tissues of new-born chicken were extracted for PCR analyzing.The results showed that pseudotyped retrovirus had transcription activity in infected CEM, and marker gene EGFP was detected in genomic DNA of 5 days chicken embryos,thymus gland and skin of one new-born chicken,which indicated technology platform of chimeric GFP transgenic chicken was built successfully.Primary study on myostatin functions in early development of chicken embryos. Four target fragments(19bp) of chicken myostatin gene coding sequences were designed and cloned into RNAi retroviral expression vector pSIREN-RetroQ. Analysis of target segments in CEM showed that transcription activity of myostatin could be blocked efficiently by RNAi. Then Pantropic RNAi VSV-G retrovirus were packaged, concentrated, and injected into blastoderm below-cavity of new hatching eggs through transgenic chicken technology.Total RNA of some five days whole embryos were analyzed by semi-quantity RT-PCR.The results showed that myostatin gene expression could be down-regulated by RNAi retrovirus in 5 days chicken embryos , while MyoD and myogenin expression was visibly up-regulated, P21 expression was down-regulated and promoted myostatin down-regulated in muscle tissue. In addition, In situ hybrization(ISH) analysis showed that myostatin expression was different in a wide wariety of tissues in embryo and highly expressed in brain and eyes. All these results suggested that myostatin might have more important functions for chicken early embryo development.Primary study on myostatin function in chickling pectoral muscle growth and development. Concentrated RNAi retrovirus and RNAi recombinant plasmids were injected into new-born chicken chest muscle with different dosage respectively, and the control group chicken were injected with PBS solution .These chicken were permitted to take food and drink water freely, and all chicken were killed mercifully for comparing pectoral muscle phenotype between different groups. At the same time, RNA and protein of each group chicken chest muscle were isolated for analyzing. The results showed that myostatin expression could be blocked efficiently by injected RNAi plasmid or retrovirus, and upregulating the transcription level of MyoD, myogenin, Pax7 and mIGF-1, and downregulating P2. The pectoral muscle of RNAi groups had bigger muscle mass and more nucleus in myofibrils than control one.To better understand the mechanism of myostatin gene expression and regulation, the activity of chicken myostatin promoter was analyzed. We cloned and characterized a 2.0-kb fragment containing the 5'-regulatory region of the broiler and layer myostatin gene. Sequences analysis indicated that myostatin promoter was highly conserved among animals of different species. The broiler and layer myostatin promoter sequences consisted of several CAAT-boxes and E-boxes, and some muscle growth response elements existed in bovine, porcine and goat etc.were detected conservely in broiler and layer. Deletion analysis according to the SNPs of broiler and layer myostatin promoter was used to measure broiler and layer myostatin promoter activity with Luciferase Assay System andβ-Galactosidase Enzyme Assay System. The six different length myostatin promoter fragments were cloned into pGL3-Basic vector and transfected into CEM of broiler and layer respectively or reciprocally. The results indicated that obvious activity difference existed between broiler and layer myostatin promoter. There was significant difference in activity at 0.2kb fragment of broiler and layer myostatin promoter. However, broiler and layer myostatin promoters had high activity in corresponding cell entironment respectively, but layer cell entironment was more suited for myostatin gene transcription than broiler. Mutation analysis showed that -19bp SNP was the key site of 0.2kb fragment, and activity difference between broiler and layer myostatin promoters. In short, there existed differences in transcription activity between broiler and layer myostatin promoter, which was related to the SNPs of the myostatin promoter sequences and different cell environment.Analysises above show that the study of myostatin functions in early chicken embryo gains better results with the help of technology of transgenic chicken platform and RNAi. The results show that myostatin affects myogenesis and the expression of related genes markedly in early chicken embryos. The resluts also show myostatin possibly has additional functions other than myogenesis. In addition, myostatin plays an important role in the chicken skeleton muscle growth and development in vivo. Promoter analysis indicates that there are some activity differences between broiler and layer myostatin promoters, and these differences are related to cell environment and promoter sequences. Our experiments focus on the study of myostatin gene structure, expression and function in chicken. We expect the data could provide references for further study on myostatin gene functions and applications.