牦牛IGF-II干扰载体的构建及其转染对睾丸支持细胞的影响

doi:10.11843/j.issn.0366-6964.2020.09.011

Abstract

Abstract: The aim of this study was to construct shRNA recombinant vector interfering insulin-like growth factor-II (IGF-II), and study the effects of IGF-II on the yak sertoli cells. The shRNA oligonucleotides targeting yak IGF-II were designed and synthesized and it was cloned into pLKO.1 plasmid vector, after transfected into yak sertoli cells, a stable cell line was obtained by puromycin selection. The relative expressions of IGF-II mRNA and protein were identified by qRT-PCR and Western blotting, respectively. The proliferation and apoptosis of sertoli cells were analyzed by proliferation measurement system. The results showed that the pLKO.1-shRNA vector interfering IGF-II was successfully constructed, which was stably expressed in sertoli cells and could interfere the expression of yak IGF-II effectively. Compared with the control group, the interference efficiency of pLKO.1-shRNA2 was the most significant， IGF-II expression down-regulated to 19.1% (P<0.05), and could reduce the expression of endogenous IGF-II protein by 76.3% (P<0.05). The stable cell line obtained by transfection and screening of pLKO.1-shRNA2 plasmid had significant lower activity of cell division and proliferation than that of control group after 48 h of culture (P<0.05). The results of qRT-PCR showed that the expressions of IGF-I and IGF-IIR in sertoli cells were significantly up-regulated (P<0.05), and the expressions of IGF-IR and Bcl-2 were significantly down-regulated (P<0.05) after interfering IGF-II. In conclusion, the interference plasmids of IGF-II were successfully constructed. After interference plasmid was transfected into yak sertoli cells, it could effectively inhibit the expression of IGF-II, change the expression pattern of IGF-IR and Bcl-2, and potentially affect the division and proliferation of yak sertoli cells, however, the specific regulation mechanism requires further studied.

Key words: yak, sertoli cells, IGF-II, interfere, cell proliferation

CLC Number:

S823.85

ZHANG Lei, AGUO Yueda, XIE Lazhun, WU Jinbo, LI Jian, XIONG Xianrong. Construction of IGF-II shRNA Knockdown Vector and Its Effect on Sertoli Cells in Yak[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(9): 2138-2146.

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Construction of IGF-II shRNA Knockdown Vector and Its Effect on Sertoli Cells in Yak

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