小鼠生精细胞特异表达载体AAV-Dazl-RFP-Flag的构建及表达验证  

Construction and expression verification of mouse spermatogenic cell-specific expression vector AAV-Dazl-RFP-Flag

作　　者：李雅惠 李凯 罗艳云[2] 李梦真 邙新雨 宋伟[2] 杨涛[1] LI Ya-hui;LI Kai;LUO Yan-yun;LI Meng-zhen;MANG Xin-yu;SONG Wei;YANG Tao(Department of Biochemistry and Molecular Biology,Shanxi Medical University,Taiyuan 030001;National Laboratory of Medical Molecular Biology,Institute of Basic Medical Sciences CAMS,School of Basic Medicine PUMC,Beijing 100005,China)

机构地区：[1]山西医科大学生物化学与分子生物学教研室,山西太原030001 [2]中国医学科学院基础医学研究所北京协和医学院基础学院医学分子生物学国家重点实验室,北京100005

出　　处：《基础医学与临床》2020年第6期753-758,共6页Basic and Clinical Medicine

基　　金：国家自然科学基金(31970794);国家重点实验室专项基金(2060204);山西省重点研发计划(201803D31072);山西省重点研发计划重点项目(201703D111029)。

摘　　要：目的构建含有生精细胞特异启动子Dazl的真核报告基因表达载体AAV-Dazl-RFP-Flag,研究生精细胞特异启动子Dazl在生精细胞中的启动效果。方法通过同源重组的方法对腺相关病毒载体AAV-CMV-RFP-Flag进行改造,构建小鼠生精细胞特异表达载体AAV-Dazl-RFP-Flag。在体外,转染GC1-spg野生型细胞和HEK-293T细胞,通过免疫荧光、q-PCR、Western blot验证AAV-Dazl-RFP-Flag在体外的表达特异性。在体内,将AAV-Dazl-RFP-Flag进行腺相关病毒(AAV)包装,通过显微注射技术将AAV注射到小鼠睾丸内,通过q-PCR检测AAV-Dazl-RFP-Flag在体内的表达特异性。结果成功构建了AAV-Dazl-RFP-Flag表达载体,并在体内、外水平验证了AAV-Dazl-RFP-Flag的表达。结论小鼠生精细胞特异表达载体AAV-Dazl-RFP-Flag可以在生精细胞中特异性表达,为雄性生殖不育的基因治疗提供更高效的载体工具。Objective To study the effect of spermatogenic cell-specific promoter Dazl in Spermatogenic cells.Constructing the eukaryotic reporter gene expression vector AAV-Dazl-RFP-Flag containing spermatogenic cell-specific promoter Dazl.Methods The Adeno-associated virus vector AAV-CMV-RFP-Flag was modified by homologous recombination to construct mouse permatogenic cell-specific expression vector AAV-Dazl-RFP-Flag.In vitro,GC1-spg wild-type cells and HEK-293 T cells were transfected,and the expression specificity of AAV-Dazl-RFP-Flag in vitro was verified by immunofluorescence,q-PCR,and Western blot.In vivo,AAV-Dazl-RFP-Flag was packaged with adeno-associated virus(AAV),and AAV was injected into the testes of mouse through microinjection technology,then the expression specificity of AAV-Dazl-RFP-Flag in vivo was detected by q-PCR.Results Mouse Sper-matogenic cell-specific expression vector AAV-Dazl-RFP-Flag is specifically expressed in spermatogenic,cells thus provid providing a more efficient tool for gene therapy of male reproductive infertility.

关 键 词：DAZL 启动子 基因治疗 精子发生 

分 类 号：R334[医药卫生—人体生理学]