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Asian Journal of Animal and Veterinary Advances
Year: 2013  |  Volume: 8  |  Issue: 4  |  Page No.: 571 - 581

Construction of Myostatin Gene Knockout Vector in Goat and Transfection Of Nuclear Donor Cells

Zi-Jun Zhang, Ying-Hui Ling, Chun-Huan Ren, Xiao Cheng, Hong-Guo Cao, Xiao-Fei Guo, Ya-Feng Huang and Xiao-Rong Zhang    

Abstract: Myostatin (MSTN) was a major gene of skeletal muscle growth regulation and a member of the transforming growth factor-β (TGF-β) super family. It acts as a negative regulator of skeletal muscle growth. To knock out goat MSTN gene of nuclear donor cells correctly, a gene targeting vector should be constructed. The homologous arm of goat MSTN was amplified by PCR and restriction enzyme digested. Intermediate vector pMD18-SA and pMD18-LA were constructed by conventional molecular cloning method. The final target vector pLOXP-MSTN was constructed by step-by-step cloning. The replacement type knock-out vector was transfected into goat fetal fibroblast by electrotransfection. The homologous arm sequences of goat MSTN including the 4.6 kb homologous long arm (LA) and the 1.9 kb homologous Short Arm (SA) were successfully amplified using LA-PCR technology. The LA and SA were inserted into vector pLOXP, generating MSTN gene targeting vector pLOXP-MSTN which contained neo and tk positive-negative-selection markers. The pLOXP-MSTN was linearized and electroporated into the nuclear donor cells. After G418 and GANC selection, fifty-eight drug resistant cell clones were screened and from which totally 3 positive clones were confirmed by PCR and DNA sequencing. The constructed gene targeting vector could efficiently target of MSTN locus and the gene knockout cell clones will be used to produce MSTN knockout goat by means of somatic cell nuclear transplantation.

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