慢病毒活体介导精原干细胞生产转基因小鼠  

Production of transgenic mice by lentivirus mediated spermatogonial stem cells in vivo

作　　者：辛颖[1] 张涛[1,2] 张优优[1] 张智英[1] 

机构地区：[1]西北农林科技大学动物科技学院,陕西杨凌712100 [2]陕西理工学院生物科学与工程学院,陕西汉中723001

出　　处：《西北农林科技大学学报（自然科学版）》2014年第6期15-20,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家"973"计划项目"iPS猪的规模化高效创建"(K301021001);国家自然科学基金项目"人工锌指转录激活因子激活猪体细胞内源多能因子诱导iPS细胞的研究"(K305021104)

摘　　要：【目的】用慢病毒在体内感染精原干细胞,建立转基因动物生产技术平台。【方法】用三质粒慢病毒载体系统转染293T细胞包装慢病毒,转染后裂解细胞,收集携带绿色荧光蛋白(EGFP)基因的慢病毒。将慢病毒液注射到小鼠曲细精管,获得F0代小鼠,制备小鼠睾丸组织切片,进行免疫双荧光检测。将F0代小鼠与野生型母鼠交配,获得F1代小鼠,PCR检测F1代是否为转基因小鼠。【结果】通过裂解细胞的方法成功制得慢病毒,将其注射到小鼠曲细精管,获得了10只F0代小鼠,经免疫双荧光检测,发现慢病毒可在活体上感染精原干细胞。8只F0代小鼠与野生型母鼠交配后共获得了41只F1代小鼠,经PCR检测,21只携带慢病毒基因片段,转基成功率为51.22%,表明慢病毒介导的基因改造是可遗传的。【结论】建立了一种操作简单、花费少、易于实施的慢病毒活体介导精原干细胞生产转基因小鼠的技术体系,该技术也可以推广到其他动物上,对于加速功能基因鉴定、疾病模型构建和家畜转基因育种等领域的研究有促进作用。【Objective】This research aimed to build a technical platform for production of transgenic animal using lentiviral infection of spermatogonial stem cells in vivo.【Method】Three lentiviral vectors were co-transfected into 293T cells,and virus particles expressing EGFP protein were harvested by cell lysis.Lentivirus was injected into seminiferous tubules of F0 mice.Lentivirus infected spermatogonial stem cells were detected through fluorescence immunofluorescence microscopy of mice testis tissue slices.F1 mice from hybridization of F0 and wild mice were detected by PCR.【Result】Ten 21 days old F0 mice were obtained by seminiferous tubule injection of lentvirus produced by cell lysis method.Fluorescence immunofluorescence microscopy showed that lentivirus could infect spermatogonial stem cells in vivo.21 out of 41 mice from hybridization of 8 treated F0 male mice and wild-type female mice were detected with lentiviral fragment.The transgenic rate was 51.22%.It indicated that lentivirus induced transgene was heritable.【Conclusion】A cost-efficient and easy to hand on technique for transgenic mice production was established.This technique can be extended to other animals,and it can facilitate the research of gene identification,construction of disease models and transgenic livestock breeding.

关 键 词：慢病毒 活体 精原干细胞 转基因小鼠 

分 类 号：Q785[生物学—分子生物学]