小鼠生精细胞特异表达载体AAV-Dazl-RFP-Flag的构建及表达验证

基础医学与临床 ›› 2020, Vol. 40 ›› Issue (6): 753-758.

小鼠生精细胞特异表达载体AAV-Dazl-RFP-Flag的构建及表达验证

李雅惠¹, 李凯², 罗艳云², 李梦真², 邙新雨², 宋伟², 杨涛^1*

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

收稿日期:2020-03-08 修回日期:2020-04-20 出版日期:2020-06-05 发布日期:2020-05-29
通讯作者: *yangtao056cn@126.com
基金资助:
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)

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

LI Ya-hui¹, LI Kai², LUO Yan-yun², LI Meng-zhen², MANG Xin-yu², SONG Wei², YANG Tao^1*

1. Department of Biochemistry and Molecular Biology, Shanxi Medical University, Taiyuan 030001;
2. National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences CAMS, School of Basic Medicine PUMC, Beijing 100005, China

Received:2020-03-08 Revised:2020-04-20 Online:2020-06-05 Published:2020-05-29
Contact: *yangtao056cn@126.com

摘要/Abstract

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。

关键词: Dazl, 启动子, 基因治疗, 精子发生

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-293T 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.

Key words: Dazl, promoter, gene therapy, spermatogenesis

中图分类号:

R334

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.

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 & Clinical Medicine, 2020, 40(6): 753-758.

参考文献

[1]Hermo L, Pelletier RM, Cyr DG, et al. Surfing the wave, cycle, life history, and genes/proteins expressed by testicular germ cells. Part 1: background to spermatogenesis, spermatogonia, and spermatocytes[J]. Microsc Res Tech, 2010, 73: 241-278.
[2]Luk AC, Chan WY, Rennert OM, et al. Long noncoding RNAs in spermatogenesis: insights from recent high-throughput transcriptome studies[J]. Reproduction, 2014, 147: R131-R141.
[3]Venables JP, Ruggiu M, Cooke HJ. The RNA-binding specificity of the mouse Dazl protein[J]. Nucleic Acids Res, 2001, 29: 2479-2483.
[4]Lin Y, Page DC. Dazl deficiency leads to embryonic arrest of germ cell development in XY C57BL/6 mice[J]. Dev Biol, 2005, 288: 309-316.
[5]Wang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery[J]. Nat Rev Drug Discov, 2019, 18: 358-378.
[6]Hauswirth WW, Aleman TS, Kaushal S, et al. Treatment of leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: short-term results of a phase I trial[J]. Hum Gene Ther, 2008, 19: 979-990.
[7]Lawlor PA, Bland RJ, Mouravlev A, et al. Efficient gene delivery and selective transduction of glial cells in the mammalian brain by AAV serotypes isolated from nonhu-man primates[J]. Mol Ther, 2009, 17: 1692-1702.
[8]Dirren E, Towne CL, Setola V, et al. Intracerebroventricular injection of adeno-associated virus 6 and 9 vectors for cell type-specific transgene expression in the spinal cord[J]. Hum Gene Ther, 2014, 25: 109-120.
[9]Ai J, He Y, Zheng M, et al. Characterization of recombinant adeno-associated viral transduction and safety profiles in cardiomyocytes[J]. Cell Physiol Biochem, 2018, 48: 1894-1900.
[10]Lu Q, Ganjawala TH, Ivanova E, et al. AAV-mediated transduction and targeting of retinal bipolar cells with improved mGluR6 promoters in rodents and primates[J]. Gene Ther, 2016, 23: 680-689.
[11]Welling M, Chen HH, Muñoz J, et al. DAZL regulates Tet1 translation in murine embryonic stem cells[J]. EMBO Rep, 2015, 16: 791-802.

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