Transgenic Sheep Of Skeletal Muscle Specific Expression Follistatin

The development of somatic cell nuclear transfer and transgenic technology has offered a new technical platform to the cultivation of transgenic new breeds. Follistatin (FST) is able to promote muscle growth and increase muscle strength, which has beco me the hotspot gene of high-yield livestock research and amyotrophy treatment in recent years. By combining somatic cell nuclear transfer technology with transgenic technology, this study is aiming to cultivate high-yield transgenic sheep. In this research muscle-specific expression FST gene eukaryotic expression vectors were constructed, and Mongolian sheep fetal fibroblast cells (SFCs) of stable transfection were obtained. By using transgenic cell clones that the exogenous gene stable integration of cells genome as nuclear donors and matured ovine oocytes as the recipients, transgenic embryos were produced by somatic cell nuclear transfer technology, and followed to produce transgenic Mongolian sheep through embryo transfer.1. Construction of muscle-specific expression FST gene eukaryotic expression vectors FST genes cDNA series were obtained by RT-PCR, pCCDS was obtained by digesting muscle- specific promoting sub-skeleton vectors includind porcine a-actin 5'region's. FST cDNA was connected with pCCDS, and then the muscle-specific expression FST gene eukaryotic expression vector-pCFCDS was constructed, and the vector was identified by enzyme digesting and PCR; By connecting FST cDNA with the synthetic promoter (SP), intermediate vector - p19T-SPF was obtained, p19T-SPF was digested to get SPF fragment. SPF was connected with skeleton vector pCDsReD2, muscle-specific expression vector - pSPFCDS was constructed, the vector was identified by enzyme degesting and PCR.2. Expression vector transfected sheep fetal fibroblast cells (SFCs)Use liposome mediated approach to transfer muscle-specific expression vectors pCFCDS and pSPFCDS into Mongolian SFCs, and use G418 and red fluorescent protein double selection markers to screen and get transgenetic cell clones. After enlarging the cultivation of transgenetic cells, the exogenous gene was analysed by PCR, select positive clones to draw a growth curve and analyze the karyotypes. The results of the appraisal showed that, the selected cells could express the target genes stably, the growth curve and karyotype analysis were basically normal, and the cells could be used as donor cells of somatic cell nuclear transfer in the follow-up work.3. Preparation of transgenic embryo and production of transgenic sheepNucleus and the first polar body were removed through micromanipulation after ovine oocytes in vitro maturation, and the transgenic cells which had been transfected pCFCDS or pSPFCDS stably were injected into the oocytes. After electrofusion, the reconstructed embryos were activated by 5μM IA23187 5min+2mM 6-DMAP for 4hr. The reconstructed embryos developed in 4-8 cell stage, were transplanted into the oviducts of the recipients which oestrus was in the same period. The in vitro maturation rates of sheep oocytes were 67%, the fusion rates were 78.2%, the cleavage rates of the reconstructed embryos were 68.6%, eight-cell stage embryo rates were 83.9%, and blastocyst rates were 22%.Transgenic embryos were transplanted into 87 recipient sheeps, of which 14 were pregnant, with the pregnancy rates of being 16.1%. Five of them were born and currently one was alive. After PCR appraisal, all of five lambs were integration of exogenous target genes,RNA of follistatin can be detected in skeletal muscle.