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目的:构建PIAS-NY基因慢病毒质粒,并将包装所得慢病毒感染小鼠精母细胞,获得稳定过表达PIAS-NY的细胞株。方法:应用PCR技术扩增目的基因,并将扩增产物插入慢病毒载体质粒pGC-FU上,对阳性克隆进行PCR筛选及测序鉴定。将重组质粒与两种辅助包装原件载体质粒共转染293T细胞,获得含慢病毒颗粒的细胞上清,用慢病毒感染小鼠精母细胞,并用Western印迹方法检测细胞中PIAS-NY蛋白的表达。结果:成功构建了pGC-FU-PIAS-NY慢病毒表达质粒,获得了稳定过表达PIAS-NY的小鼠精母细胞株。结论:PIAS-NY过表达慢病毒质粒及稳定转染小鼠精母细胞株的构建,为进一步体外研究PIAS-NY基因在精子发生中的功能奠定了基础。
OBJECTIVE: To construct the lentivirus plasmid of PIAS-NY gene and infect lentivirus-infected mouse spermatocytes to obtain a stable cell line stably overexpressing PIAS-NY. Methods: PCR was used to amplify the target gene. The amplified product was inserted into lentiviral plasmid pGC-FU. The positive clones were screened by PCR and sequenced. 293T cells were cotransfected with the recombinant plasmids and two kinds of helper plasmid vectors to obtain the cell supernatant containing the lentivirus particles. The mouse spermatocytes were infected with lentivirus, and the expression of PIAS-NY protein in the cells was detected by Western blotting . Results: The lentiviral vector pGC-FU-PIAS-NY was successfully constructed and the mouse spermatocyte strain stably overexpressed PIAS-NY was obtained. CONCLUSION: The construction of PIAS-NY overexpression lentiviral plasmid and stable transfected mouse spermatocyte line will lay the foundation for further study on the function of PIAS-NY gene in spermatogenesis.