基于四环素调控系统的ApoE-rtTA/TRE-HCV-C双转基因小鼠的制备

目的为了丰富现有的四环素调控系统的小鼠资源库,同时也为更好地研究目的基因HCV-C的作用机制提供一个活体的动物模型,制备基于四环素调控系统原理的调控部分ApoE-rtTA的转基因小鼠,与反应部分TRE-HCV-C转基因小鼠,相互交配制作出双转基因小鼠。方法应用显微注射法将构建好的两种基因片段ApoE-rtTA及TRE-HCV-C分别注入超排的昆明母鼠的受精卵,再植入假母输卵管,出生动物及其后代经PCR初步筛选出阳性,将两者及阳性后代交配产生携带5只转基因鼠再经Western blot和RT-PCR在RNA和蛋白水平上进一步鉴定。结果产生了3只整合有ApoE-rtTA基因的首见鼠和125只阳性子代,以及产生了5只整合有TRE-HCV-C基因的首见鼠和16只阳性子代。PCR及Southern Blot证实上述阳性鼠确有转基因整合。将两者交配产生携带5只双转基因鼠。结论成功制备了携带有ApoE-rtTA及TRE-HCV-C转基因小鼠,建立了分别携带有这两种基因的鼠群,以及同时携带有两种基因的双转基因小鼠,为四环素调控系统提供了一个良好的通用型的调控动物模型,同时也可利用四环素调控系统来研究HCV中的C基因对小鼠的作用,也是HCV-C基因功能研究及与肝细胞癌的关系的机制研究的一个有用工具。

Development of ApoE-rtTA/TRE-HCV-C Double Transgenic Mice via Tet-on System

Objective In order to enrich the present Tet mice animal bank and provide an useful tool for studies on pathogenesis of HCV-C gene,a study on establishment of a double transgenic mouse model containing both ApoE-rtTA and TRE-HCV-C elements was carried out.Method Two constructed fragments carrying ApoE-rtTA and TRE-HCVC were individually introduced into fertilized eggs by microinjection.The injected eggs were implanted into the oviducts of female mice,from them offsprings were obtained.Grossed the two strains double transgenic mice were got and identified with RT-PCR and western blot.Results Three mice carrying ApoE-rtTA gene and five mice carrying HCV-C gene were identified by PCR and Southern blotting.Double transgenic mice were got by crossing the two transgenic mice.It turned out 3 transgenic mice carrying ApoE-rtTA and 125 positive offsprings,and also 5 transgenic mice carrying TRE-HCV-C and 16 positive offsprings.Five double transgenic mice were generated.Conclusions Transgenic mice carrying tet-on systems have been developed,which enriched the world "Tet-mouse" bank,and provides an animal model for research on HCV core protein.Moreover,an ideal model is established for studies on some genes related to hepatic disease in vivo and provides a technical platform for research on gene expression with temporal,quantitative and site-speciality regulation.