PIAS-NY稳定转染小鼠精母细胞株的构建与鉴定

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。

Construction and identification of a mouse spermatocyte-derived cell line with a stable expression of PIAS-NY

Objective: To construct a lentiviral expression vector of the PIAS-NY gene,and establish a mouse spermatocyte-derived cell line with a stable overexpression of PIAS-NY.Methods: PIAS-NY was synthesized,amplified by PCR and cloned into the lentiviral vector expression plasmid pGC-FU.After digestion and sequencing,pGC-FU-PIAS-NY,pHelper 1.0 and pHelper 2.0 were co-transfected into 293T cells.Then the lentiviral particles were used to transfect the mouse spermatocyte-derived cells.The expression of the PIAS-NY protein was detected by Western blot.Results: We successfully constructed the lentiviral expression vector pGC-FU-PIAS-NY and established a mouse spermatocyte-derived cell line with a stable overexpression of PIAS-NY.Conclusion: The construction of the lentiviral expression vector pGC-FU-PIAS-NY and the obtainment of stably transfected mouse spermatocyte-derived cells have paved the way for further studies on the roles of the PIAS-NY gene in spermatogenesis.