可控性Speedy A基因表达转基因小鼠模型的建立鉴定和表型分析  

Establishment and phenotypic analysis of transgenic mice model with Speedy A controllable expression

作　　者：李媛媛[1,2] 张瑶楠 闫丽[1,2] 贾孟春 刘美玲 

机构地区：[1]北京协和医学院研究生院,北京100730 [2]国家卫生计生委科学技术研究所国家卫生计生委男性生殖健康重点实验室,北京100081

出　　处：《生殖医学杂志》2016年第7期639-645,共7页Journal of Reproductive Medicine

基　　金：国家自然科学基金面上项目(81170616);国家自然科学基金青年基金(31201116)

摘　　要：目的建立基于Tet-On系统诱导表达的Speedy A基因RNA干扰转基因小鼠模型,探讨Tet-On系统在研究精子发生相关基因中的应用,研究Speedy A基因下调对精子发生的影响。方法体外实验筛选下调Speedy A基因表达的有效干扰片段,Gateway技术构建包含有效干扰片段和四环素反应元件(TRE)的质粒(pRP.EX2d-TRE>shSpdya),受精卵显微注射该质粒建立转基因小鼠并筛选到纯合子,然后与四环素调控的反式激活子(rtTA)转基因小鼠杂交,筛选TRE和rtTA双阳性的小鼠为四环素诱导的Speedy A-RNA干扰转基因小鼠模型,喂食强力霉素(Dox)诱导RNA干扰片段的表达下调Speedy A基因的表达;实时荧光定量PCR法检测模型组小鼠与喂食强力霉素的正常小鼠(对照组)Speedy A的表达差异;HE染色观察睾丸组织形态学变化,TUNEL法检测小鼠睾丸组织的细胞凋亡情况。结果模型组小鼠Speedy A基因的表达量与对照组小鼠的表达量相比下降26%;模型组小鼠睾丸组织发生异常的生精细胞凋亡。结论成功建立了基于Tet-On系统诱导表达的可控性Speedy A-RNA干扰转基因小鼠模型,说明可以基于Tet-On系统在体研究精子发生相关基因的功能;Speedy A基因表达下调可使生精细胞发生异常凋亡。Objective： To establish transgenic mice model with Speedy A-RNAi controllable expression based on Tet-On system, and investigate the application of Tet-On system on researching the genes related with spermatogenesis,and the effects of Speedy A knockdown on spermatogenesis. Methods： The effective interference fragments of Speedy A for vector（pRP. EX2d-TRE〉shSpdya） construction which contains tetracycline-responsive element and the interference fragment were selected in vitro. The transgenic mice model with Speedy A-RNAi was obtained by micro-injecting the vector of pRP. EX2d-TRE〉shSpdya which was established by Gateway technology. The homogeneous mice were selected and mated with reverse tet-controlled transactivator transgenic mice which regulated by tetracycline. Then the mice were fed with doxycycline （Dox） to induce down-regulated expression of Speedy A by mean of induction of the expression of RNA interference fragment. The differential expression between model group and control group were detected by real-time fluorescent quantitative PCR. The morphological changes in testis were detected by HE, and apoptosis in testicular tissue by TUNNEL. Results： Compared with the control group,the expression level of Speedy A in model group decreased significantly by 26 %, and a large number of spermatogenic cell apoptosis occurred in the mouse testis of model group. Conclusions： The transgenic mice model with Speedy A-RNAi controllable expression based on Tet On system has been established. This means that the biological function of spermatogenesis related gene can be investigated in vivo based on Tet On system. The down-regulated expression of Speedy A causes abnormal apoptosis of spermatogenic cells.

关 键 词：TET-ON系统 SPEEDY A基因 RNA干扰 精子发生 

分 类 号：R[医药卫生]