牦牛lncRNAENSBGRT00000000387.1慢病毒载体构建及其对卵泡GCs凋亡的影响

doi:10.11843/j.issn.0366-6964.2023.03.019

Abstract

Abstract: The purpose of this study was to construct yak lncRNA ENSBGRT00000000387.1 overexpression lentivirus vector and infect yak follicular granulosa cells (GCs) to understand its effect on cell apoptosis. The yak follicular RNA was extracted, and the full-length sequence of lncRNA ENSBGRT00000000387.1 was amplified by RT-PCR, and then its recombinant plasmid was constructed. After it was packaged with lentivirus packaging system (pVSVG: pMDL: pRev), the infective titer on 293T cells was determined by qPCR, and then the isolated and cultured yak GCs were infected. The expression level of lncRNA ENSBGRT00000000387.1 in GCs was detected by qPCR, and the apoptosis rate of GCs was detected by flow cytometry. Western blot and qPCR were used to detect the mRNA and protein expression levels of pro-apoptotic gene CASPASE3, BAX and anti-apoptotic gene BCL-2, respectively. The full-length sequence of 2 566 bp of lncRNA ENSBGRT00000000387.1 was successfully amplified from yak follicles, and it was homologously recombined with the fragment of the homologous region of LV-EF1a-EGFP-2A vector. The restriction enzyme digestion identification and sequencing verification showed that the recombinant plasmid was successfully constructed. After it was packaged and concentrated in the lentivirus packaging system, the infection efficiency in 293T cells reached (75.77±0.850) %. The qPCR test results showed that the titer of the lentivirus was 7.32×10⁸ TU·mL^-1, indicating that the target lncRNA overexpression lentivirus vector was successfully constructed. The infection efficiency of yak follicular GCs infected with lentivirus was (52.93±1.168)%. The qPCR results showed that the expression level of target lncRNA in infected GCs was significantly increased (P<0.01). Flow cytometry showed that the apoptosis rate of cells in lentivirus infection group decreased significantly (P<0.01), the expression of pro-apoptotic genes CASPASE 3 and BAX at mRNA and protein levels decreased significantly (P<0.01), and the expression of anti-apoptotic gene BCL-2 increased significantly (P<0.01). In this experiment, lncRNA ENSBGRT00000000387.1 lentivirus vector was successfully constructed. After successfully infecting yak follicular GCs, it was found to have the effect of inhibiting apoptosis, indicating that it may play a role in yak follicular function.

Key words: lncRNA, lentivirus vector, granulosa cells, apoptosis, yak

CLC Number:

S823.85

MENG Zhaoyi, WANG Yunlu, XU Yefen, NIU Jiaqiang, SUOLANG Sizhu, GUO Min, XI Guangyin. Construction of Yak lncRNA ENSBGRT00000000387.1 Lentivirus Vector and Its Effect on Apoptosis of Yak Follicular Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 1058-1070.

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Construction of Yak lncRNA ENSBGRT00000000387.1 Lentivirus Vector and Its Effect on Apoptosis of Yak Follicular Granulosa Cells

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