An efficient strategy for generation of transgenic mice by lentiviral transduction of male germline stem cells in vivo  

作　　者：Jinzhou Qin Haixia Xu Pengfei Zhang Conghui Zhang Zhendong Zhu Rongfeng Qu Yuwei Qin Wenxian Zeng 

机构地区：[1]College of Animal Science and Technology, Northwest A&F University

出　　处：《Journal of Animal Science and Biotechnology》2016年第2期158-166,共9页畜牧与生物技术杂志（英文版）

基　　金：supported in part by National Basic Research Program of China (973 program; 2013CB943103);the National Natural Science Foundation of China (Grant No. 31072029, No.31272439, No. C170104 and No. 31230048);Ph.D. Programs Foundation of Ministry of Education of China (Grant No.20130204110017) for W. Zeng and W Dong;the scholarship from China Scholarship Council (CSC)

摘　　要：Background： Male germline stem cells（MGSCs） are a subpopulation of germ cells in the testis tissue. MGSCs are capable of differentiation into spermatozoa and thus are perfect targets for genomic manipulation to generate transgenic animals.Method： The present study was to optimize a protocol of production of transgenic mice through transduction of MGSCs in vivo using lentiviral-based vectors. The recombinant lentiviral vectors with either EF-1 or CMV promoter to drive the expression of enhanced green fluorescent protein（e GFP） transgene were injected into seminiferous tubules or inter-tubular space of 7-day-old and 28-day-old mouse testes. At 5 or 6 wk post-surgery, these pre-founders were mated with wild-type C57BL/6J female mice（1.5 to 2.0-month-old）.Results： Sixty-seven percent of F1 generation and 55.56 % of F2 offspring were positive for eG FP transgene under the control of EF-1 promoter via PCR analysis. The transgenic pups were generated in an injection site-and age-independent manner. The expression of transgene was displayed in the progeny derived from lentiviral vector containing CMV promoter to drive transgene, but it was silenced or undetectable in the offspring derived from lentiviral vector with transgene under EF-1 promoter. The methylation level of g DNA in the promoter region of transgene was much higher in the samples derived lentiviral vectors with EF-1 promoter than that with CMV promoter,suggesting e GFP transgene was suppressed by DNA methylation in vivo.Conclusion： This research reported here an effective strategy for generation of transgenic mice through transduction of MGSCs in vivo using lentivirus vectors with specific promoters, and the transgenic offspring were obtained in an injection site-and age-independent manner. This protocol could be applied to other animal species, leading to advancement of animal transgenesis in agricultural and biomedical fields.Background： Male germline stem cells（MGSCs） are a subpopulation of germ cells in the testis tissue. MGSCs are capable of differentiation into spermatozoa and thus are perfect targets for genomic manipulation to generate transgenic animals.Method： The present study was to optimize a protocol of production of transgenic mice through transduction of MGSCs in vivo using lentiviral-based vectors. The recombinant lentiviral vectors with either EF-1 or CMV promoter to drive the expression of enhanced green fluorescent protein（e GFP） transgene were injected into seminiferous tubules or inter-tubular space of 7-day-old and 28-day-old mouse testes. At 5 or 6 wk post-surgery, these pre-founders were mated with wild-type C57BL/6J female mice（1.5 to 2.0-month-old）.Results： Sixty-seven percent of F1 generation and 55.56 % of F2 offspring were positive for eG FP transgene under the control of EF-1 promoter via PCR analysis. The transgenic pups were generated in an injection site-and age-independent manner. The expression of transgene was displayed in the progeny derived from lentiviral vector containing CMV promoter to drive transgene, but it was silenced or undetectable in the offspring derived from lentiviral vector with transgene under EF-1 promoter. The methylation level of g DNA in the promoter region of transgene was much higher in the samples derived lentiviral vectors with EF-1 promoter than that with CMV promoter,suggesting e GFP transgene was suppressed by DNA methylation in vivo.Conclusion： This research reported here an effective strategy for generation of transgenic mice through transduction of MGSCs in vivo using lentivirus vectors with specific promoters, and the transgenic offspring were obtained in an injection site-and age-independent manner. This protocol could be applied to other animal species, leading to advancement of animal transgenesis in agricultural and biomedical fields.

关 键 词：In vivo Lentivirus vectors Male germline stem cells Transgenesis 

分 类 号：S865.13[农业科学—野生动物驯养]