Scientia Agricultura Sinica ›› 2013, Vol. 46 ›› Issue (14): 3032-3039.doi: 10.3864/j.issn.0578-1752.2013.14.019

• ANIMAL SCIENCE·VETERINARY SCIENCERE·SOURCE INSECT • Previous Articles     Next Articles

Construction of MyoD1 Expressed Goat Embryonic Fibroblast Line and the Myogenesis of This Cell

 LI  Wei, ZHENG  Meng-Meng, ZHANG  Ya-Ni, ZHU  Cai-Ye, QIU  Feng-Long, WEI  Guang-Hui, LI  Bi-Chun   

  1. The College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, Jiangsu
  • Received:2013-02-21 Online:2013-07-15 Published:2013-05-13

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Abstract: 【Objective】 MyoD1 ectopic expression goat fibroblast line was constructed to explore the bio-functions of MyoD1 in myogenesis. 【Method】 MyoD1 was cloned from the skeletal satellite cell via RT-PCR method. To visualize the MyoD1 protein, MyoD1 was reconstructed to pEGFP-N1 with the mutation of TGA (termination codon) into GGA (glycine) and cloned into pEGFP-N1, a fusion protein expression vector. The MyoD1 expressed goat embryonic fibroblast (GEF) was founded to explore the function of MyoD1 during the myogenesis, via transfecting the pEGFP-MyoD vector into GEF with the help of LipofectiminTM LTX. MyoD1 expressed GEF was induced to undergo the myogenesis by the differentiation media. The myogenesis of this cell line was ditermined by morphology and indirect immunofluorescence assay (IFA). 【Result】 The sequencing results indicated that MyoD1 was successfully reconstructed into pEGFP-N1 with mutation of TGA into GGA, and MyoD1 was fused with eGFP. MyoD1 expressed GEF line was obtained through two weeks of continuous G418 resistance screening and expressed Myf5 (detected by indirect immunofluorescence assay (IFA)). A few signs of myotube were observed 2 days after differentiation treatment, which expressed the differentiation marker of myoblast (MyoG, Desmin, MyHC), while lots of myotube were obtained with contractility. 【Conclusion】MyoD1 gene was cloned and reconstructed with pEGFG-N1 to obtain pEGFP-MyoD1 expressed GEF line, successfully. The expression of MyoD1 would drive the GEF into myoblast with differentiation ability of myogenesis.

Key words: MyoD1 gene , gene clone , ectopic expression , myogenesis

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