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

Abstract

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

CLC Number:

R334

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.

References

[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.

[1]	LIU Jian. Progress of nucleic acid drugs and non-viral carriers in gene therapy [J]. Basic & Clinical Medicine, 2022, 42(1): 41-50.
[2]	ZHONG Hong-li, XIAO Cheng-liang, SHI Xiang-rui, DAI Yu-jie, LIU Wei, ZHANG Qing-hua. Expression, activity measurement and structure prediction of spermatogenic regulator:recombinant human HSF5 [J]. Basic & Clinical Medicine, 2021, 41(9): 1323-1328.
[3]	LI Kai, MANG Xin-yu, ZOU Ding-feng, LI Meng-zhen, MIAO Shi-ying, WANG Lin-fang, SONG Wei. Dynamic expression of RNA-binding proteins in spermatogenesis based on RNA-seq [J]. Basic & Clinical Medicine, 2021, 41(6): 825-830.
[4]	YANG Xiao-juan, LI Zhen-hao, GOU Yuan-feng, HUO Xia-qin, PEI Ya-ping, LI Na, LIU Hui-ling. Advances in adeno-associated virus mediated gene therapy in tumors [J]. Basic & Clinical Medicine, 2021, 41(4): 573-577.
[5]	XU Han-yuan, ZHU Hui-juan, PAN Hui, YANG Hong-bo, WANG Lin-jie, GONG Feng-ying. Effects of adiponectin on the gene promoter activities of fatty acid synthase and hormone sensitive lipase in HepG2 cells [J]. Basic & Clinical Medicine, 2021, 41(1): 13-19.
[6]	HU Jing-cen, LI Lian, WANG Shu-yu, XU Lei, YANG De-lun, HAN Li-yuan, XU Guo-dong. Association between SHMT1 gene promoter methylation and ischemic stroke [J]. Basic & Clinical Medicine, 2020, 40(4): 451-455.
[7]	. Progress of CRISPR/Cas9 gene editing technology in cardiovascular field [J]. Basic & Clinical Medicine, 2019, 39(6): 890-894.
[8]	. Construction and characterization of QKI gene knockout GC1-spg cell strain with CRISPR/CAS9 [J]. Basic & Clinical Medicine, 2018, 38(5): 589-593.
[9]	. Advances in research on anti-Mullerian tube hormone and male reproductive endocrine-related diseases [J]. Basic & Clinical Medicine, 2018, 38(3): 405-408.
[10]	. Up-regulated expression of NT-3 attenuates cerebral ischemia/reperfusion injury in rats [J]. Basic & Clinical Medicine, 2015, 35(9): 1199-1204.
[11]	. LNAzyme specific targeting against to both 5ˊ-NCR and C gene expression of hepatitis C virus [J]. Basic & Clinical Medicine, 2015, 35(7): 938-942.
[12]	. Hepatitis B virus(HBV) S gene-specific anti-gene locked nucleic acid(LNA) significantly inhibits S gene expression in vitro [J]. Basic & Clinical Medicine, 2014, 34(2): 206-210.
[13]	. Effects of AQP1 knock down by RNAi on morphology and water transport in mouse Schwann cells [J]. Basic & Clinical Medicine, 2014, 34(1): 29-35.
[14]	. Hepatitis B virus(HBV) preS1 gene-specific anti-gene locked nucleic acid(LNA) significantly inhibits preS1 gene expression in vitro [J]. Basic & Clinical Medicine, 2013, 33(6): 722-725.
[15]	. Construction of hGHp-regulating TNFα-expression vector and its therapeutic effect on in vitro pituitary growth hormone adenoma [J]. Basic & Clinical Medicine, 2013, 33(5): 572-577.