腺病毒介导shRNA干扰绵羊MSTN基因效果及对生肌调节因子和干扰素反应基因表达的影响

doi:10.11843/j.issn.0366-6964.2017.10.006

Abstract

Abstract:

The aim of this study was to further reveal the regulation mechanism of MSTN in sheep myoblasts, prepare the tool silencing MSTN, and provide the methods and theoretical basis for increasing the yield of muscle mass by using RNA interference technology. In this study, sheep myoblasts were used as experimental materials. The short hairpin RNA(shRNA) expression plasmid vector specific targeting to MSTN gene were constructed, then the plasmids with better interference effect were packaged for recombinant adenovirus, the expression of MSTN, myogenic regulatory factor and interferon response genes in myoblasts after infected by recombinant adenovirus were detected by qRT-PCR and Western blot. The result showed that the interference efficiency of plasmid ShR218, ShR511 were 35% and 48%,respectively,the interference efficiency of ShR3+4 was 85%.Recombinant adenovirus vector Sh511 and Sh3+4 were successfully packaged, the virus titer reached 1×10⁸ pfu·mL^-1, the infection rate to myoblasts reached more than 90%. The mRNA expression of MSTN gene had been decreased by 53% and 76%, and the protein level had been decreased by 55% and 64% through Sh511 and Sh3+4, respectively. The knockdown of MSTN gene was accompanied with expression downregulation of myogenic regulatory factor Myf5, MyoD, MyoG and Myf6 at mRNA level(P<0.01), but at protein level only MyoG was significantly increased(P<0.01), no significant changes in Myf5, MyoD and Myf6. Adenovirus infecting myoblasts did not cause significant changes at OAS1 mRNA level, but caused significant increase at IFNGR1 mRNA level (P<0.01), while had no significant effect on their protein levels. In this study, shRNA adenovirus vector targeting to MSTN gene was successfully constructed, which can effectively inhibit the mRNA and protein expression of MSTN in myoblasts, and affect the expression of Myf5, MyoD, MyoG, Myf6 and IFNGR1 genes.

CLC Number:

S826.2

WANG Hong-na, SUN Hong-xin, ZHANG Ying-jie, LIU Yue-qin, GU Zhen-hui, WANG Su-yao. Adenovirus-mediated shRNA Interference of MSTN Gene and Its Effect on Expression of Myogenic Regulatory Factor and Interferon Response Gene in Sheep[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(10): 1833-1842.

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Adenovirus-mediated shRNA Interference of MSTN Gene and Its Effect on Expression of Myogenic Regulatory Factor and Interferon Response Gene in Sheep

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