| 小鼠生精细胞特异表达载体AAV-Dazl-RFP-Flag的构建及表达验证 |
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| 引用本文： | 李雅惠,李凯,罗艳云,李梦真,邙新雨,宋伟,杨涛.小鼠生精细胞特异表达载体AAV-Dazl-RFP-Flag的构建及表达验证[J].基础医学与临床,2020,40(6):753-758. |
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| 作者姓名： | 李雅惠 李凯 罗艳云 李梦真 邙新雨 宋伟 杨涛 |
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| 作者单位： | 山西医科大学 生物化学与分子生物学教研室,山西 太原030001;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 |
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| 基金项目： | 国家自然科学基金;山西省重点研发计划;国家重点实验室基金;山西省重点研发计划重点项目 |
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| 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。
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| 关 键 词： | DAZL 启动子 基因治疗 精子发生 |
Construction and expression verification of mouse spermatogenic cell-specific expression vector AAV-Dazl-RFP-Flag |
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| LI Ya-hui,LI Kai,LUO Yan-yun,LI Meng-zhen,MANG Xin-yu,SONG Wei,YANG Tao.Construction and expression verification of mouse spermatogenic cell-specific expression vector AAV-Dazl-RFP-Flag[J].Basic Medical Sciences and Clinics,2020,40(6):753-758. |
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| Authors: | LI Ya-hui LI Kai LUO Yan-yun LI Meng-zhen MANG Xin-yu SONG Wei YANG Tao |
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| Institution: | (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) |
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| Abstract: | 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. |
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| Keywords: | Dazl promoter gene therapy spermatogenesis |
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