VRTN Promoter-hsp68-LacZ表达载体的构建与鉴定

Construction and Identificationof VRTN Promoter-hsp68-LacZ Expression Vector

利用β-半乳糖苷酶(LacZ)报告基因对与猪脊椎发育相关的VRTN基因调节突变区进行研究和鉴别。使用通路克隆技术构建与VRTN基因的启动子区与热休克蛋白(hsp68)启动子及LacZ相连的报告载体,将其转染人肾上皮细胞系(HEK293T细胞),并将载体线性化后整合入HEK293T细胞基因组中进行上述表达质粒整合效率的评估。结果表明:利用通路克隆技术构建真核表达质粒快速准确;在脂质体介导下转染HEK293T细胞后,将细胞置于42℃热休克染色后检测转染效率60%左右;线性化后转染HEK293T细胞加新霉素G418筛选后可得到稳定遗传的整合有质粒的细胞。

In this study,β-galactosidase（ LacZ） reporter gene was used to explore and identify the regulatory mutations of the VRTN gene which is associated with the spine development of swine.Here,a gateway compatible report vector that contains the promoter of VRTN gene and heat shock protein（ hsp68） promoter coupled to the β-galactosidase（ LacZ） reporter gene was constructed by gateway cloning technology.Circle or linearized vector was transfected into human kidney epithelial cells（ HEK293T）.To evaluate integration efficiency of linearized vector,the number of G418-resistant colony formation was measured after the cells grew in medium supplemented with G418 for 1 week.The results show that gateway cloning technology is quick and accurate to construct VRTN Promoter-hsp68-Lac Zreporter plasmid; after Lac Z stain,the blue cell dots were observed in60% HEK293 T cells after 42 ℃ heat shock and some G418-resistant colonies indicated that the linearized vectors integrated into HEK293 T cell genome.