| Myogenin (MyoG) is a member of myogenic regulatory factors (MRFs), belonging to the bHLH protein family. MyoG is mainly involved in regulating the fusion of myotube, showing irreplaceable significance in myogenesis during embryogenesis. Transfection of MyoG into the non-muscle cells can produce cells expressing muscle-specific markers and finally results in myogenesis. As a member of MRFs, MyoG gene is the only factor expressing in all skeletal muscle cell lines. Currently, studies about cloning, expression and fuction of this gene mainly focused on model organisms, such as mouse, rat and zebra fish. Study about sheep MyoG biological activity of has not been reported. In this study, MyoG gene of sheep was cloned, and its eukaryotic expression vectors were constructed. Then the expression of MyoG gene in vitro was ahieved and subcellular localization of the expression product was analyzed by fluorescence microscopy. The study has also detected the MyoG myogenic activity and produced transgenic sheep through SSCs transfected in vivo. Major contents in this study are as follows:1. Cloning and sequence analysis of MyoG. Three exons of MyoG gene were amplified from Hu Sheep genomic DNA, then linked by overlapping extention PCR, and inserted to pMD19-T vector. Restriction enzyme digestion and sequencing confirmed that the recombinant vector pMD19-T-MyoG was correctly constructed, and the MyoG gene ORF was correct. Sequence alignment showed its homology with MyoG cDNA from GenBank FJ607135, NM-001111325. NM-001012406, NM-131189was up to99.26%,97.04%,92.00%,87.70%, and there was typical bHLH structure in the protein. These data indicate that the full-length MyoG cDNA was cloned correctly, which will play a great role in the following research.2. Expression of MyoG in vitro and subcellular location of its expression product. Recombinant eukaryotic expression vector pEGFP-Cl-MyoG was constructed. After identification, the vector was transfected into NIH-3T3cells, chicken embryo fibroblasts (CEF), goat embryo fibroblasts (GEF).48h after transfection the transfected cells were observed under fluorescence microscopy. The expression product was detected by RT-PCR and Western Blot. The results showed MyoG can express in NIH-3T3cells and the EGFP-MyoG fusion protein located at the nucleus in each kind of cell, indicating that MyoG was nucleus protein.3. Study on myogenic specificity of MyoG. To detect its myogenic activity, MyoG cDNA was inserted into the pcDNA3.0vector. Then the recombinant plasmid pcDNA3.0-MyoG was transfected into the GEF using liposomes-mediated method, and the transfected cells were selected by G418to obtain positive cell lines. After expanding culture. Indirect Fluorescence Assay (IFA) can detect the expression of Desmin. And the positive cells could fuse into myotubes. These data indicated the MyoG could convert GEF to myoblasts.4. Study on transgenic sheep mediated by SSCs. As target exogenous DNA, pEGFP-C1-MyoG was injected to the testes of sheep and goat to transfect SSCs in vivo in order to produce transgenic animals. PCR and DNA dot blot showed that the exogenous DNA has been integrated into the F1offspring genome.25.6%(7/27) goat offspring and17.9%(5/28) sheep offspring carried the recombinant vector gene. Western Blot data showed2goats and2sheep can express the exogenous gene. The data above suggested that testis-mediated gene transfer is feasible method for the generation of transgenic animal. |
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