m6A识别蛋白Ythdf3在精子发生中的作用研究
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摘要
目的:通过基因敲除模型研究N6-甲基腺嘌呤(m6A)识别蛋白Ythdf3在小鼠精子发生过程的调控作用。方法:利用CRISPR/Cas9技术构建Ythdf3基因敲除小鼠,通过免疫荧光和HE染色对敲除小鼠进行表型分析,研究Ythdf3在调控小鼠精子发生过程中的作用。结果:免疫组化染色显示Ythdf3在精原及各级生精细胞核中均有表达;成功构建Ythdf3基因敲除小鼠模型;HE染色显示Ythdf3敲除小鼠睾丸各级生精时相及附睾尾精子与对照相比无明显异常。结论:在正常生理条件下,敲除Ythdf3不影响雄性小鼠精子发生。
Objective:This study aims to investigate the role of N6-methyladenosine(m6 A)reader protein Ythdf3 during spermatogenesis in knockout mouse model. Methods:We generated Ythdf3 knockout mice by CRISPR/Cas9 technology,and detected the role of Ythdf3 in spermatogenesis by immunofluorescence,HE staining. Results:Immunohistochemistry results showed that Ythdf3 expressed in the nuclear of spermatogonia and spermatocytes;Ythdf3 knockout mice were constructed successfully;HE staining results revealed spermatogenic wave and the morphology of epididymis cauda in Ythdf3 knockout mice were normal compared with control.Conclusion:Depletion of Ythdf3 did not affect mouse spermatogenesis in normal physiological condition.
Objective:This study aims to investigate the role of N6-methyladenosine(m6 A)reader protein Ythdf3 during spermatogenesis in knockout mouse model. Methods:We generated Ythdf3 knockout mice by CRISPR/Cas9 technology,and detected the role of Ythdf3 in spermatogenesis by immunofluorescence,HE staining. Results:Immunohistochemistry results showed that Ythdf3 expressed in the nuclear of spermatogonia and spermatocytes;Ythdf3 knockout mice were constructed successfully;HE staining results revealed spermatogenic wave and the morphology of epididymis cauda in Ythdf3 knockout mice were normal compared with control.Conclusion:Depletion of Ythdf3 did not affect mouse spermatogenesis in normal physiological condition.
引文
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[10]刘媛媛,宋小玲,王仿竹. m6A识别蛋白Ythdf1在精子发生中的作用研究[J].南京医科大学学报(自然科学版),2019,39(2):171-175
[11]Ivanova I,Much C,Di Giacomo M,et al. The RNA m6A reader YTHDF2 is essential for the post-transcriptional regulation of the maternal transcriptome and oocyte competence[J]. Mol Cell,2017,67(6):1059-1067
[12]Shi H,Wang X,Lu Z,et al. YTHDF3 facilitates translation and decay of N6-methyladenosine-modified RNA[J].Cell Res,2017,27(3):315-328
[13]Li A,Chen YS,Ping XL,et al. Cytoplasmic m6A reader YTHDF3 promotes mRNA translation[J]. Cell Res,2017,27(3):444-447
[14]Shen B,Zhang W,Zhang J,et al. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects[J]. Nat Methods,2014,11(4):399-402
[15]da Cruz I,Rodríguez-Casuriaga R,Santi?aque FF,et al.Transcriptome analysis of highly purified mouse spermatogenic cell populations:gene expression signatures switch from meiotic-to postmeiotic-related processes at pachytene stage[J]. BMC Genomics,2016,17:294
[16]Zhou J,Wan J,Gao X,et al. Dynamic m(6)A mRNA methylation directs translational control of heat shock response[J]. Nature,2015,526(7574):591-594
[2] Meyer KD,Saletore Y,Zumbo P,et al. Comprehensive analysis of mRNA methylation reveals enrichment in 3’UTRs and near stop codons[J]. Cell,2012,149(7):1635-1646
[3] Wu RF,Jiang DH,Wang YZ,et al. N6-methyladenosine(m6A)methylation in mRNA with a dynamic and reversible epigenetic modification[J]. Mol Biotechnol,2016,58(7):450-459
[4] Yang Y,Hsu PJ,Chen YS,et al. Dynamic transcriptomic m(6)A decoration:writers,erasers,readers and functions in RNA metabolism[J]. Cell Res,2018,28(6):616-624
[5] Xu K,Yang Y,Feng GH,et al. Mettl3 mediated m(6)A regulates spermatogonial differentiationand meiosis initiation[J]. Cell Res,2017,27(9):1100-1114
[6] Lin Z,Hsu PJ,Xing X,et al. Mettl3/Mettl14-mediated mRNA N6-methyladenosine modulates murine spermatogenesis[J]. Cell Res,2017,27(10):1216-1230
[7] Tang C,Klukovich R,Peng H,et al. ALKBH5-dependent m6A demethylation controls splicing and stability of long3’-UTR mRNAs in male germ cells[J]. Proc Natl Acad Sci USA,2018,115(2):E325-E333
[8] Kasowitz SD,Ma J,Anderson SJ,et al. Nuclear m6A reader YTHDC1 regulates alternative polyadenylation and splicing during mouse oocyte development[J]. PLoS Genet,2018,14(5):e1007412
[9] Hsu PJ,Zhu Y,Ma H,et al. Ythdc2 is an N(6)-methyladenosine binding protein that regulates mammalian spermatogenesis[J]. Cell Res,2017,27(9):1115-1127
[10]刘媛媛,宋小玲,王仿竹. m6A识别蛋白Ythdf1在精子发生中的作用研究[J].南京医科大学学报(自然科学版),2019,39(2):171-175
[11]Ivanova I,Much C,Di Giacomo M,et al. The RNA m6A reader YTHDF2 is essential for the post-transcriptional regulation of the maternal transcriptome and oocyte competence[J]. Mol Cell,2017,67(6):1059-1067
[12]Shi H,Wang X,Lu Z,et al. YTHDF3 facilitates translation and decay of N6-methyladenosine-modified RNA[J].Cell Res,2017,27(3):315-328
[13]Li A,Chen YS,Ping XL,et al. Cytoplasmic m6A reader YTHDF3 promotes mRNA translation[J]. Cell Res,2017,27(3):444-447
[14]Shen B,Zhang W,Zhang J,et al. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects[J]. Nat Methods,2014,11(4):399-402
[15]da Cruz I,Rodríguez-Casuriaga R,Santi?aque FF,et al.Transcriptome analysis of highly purified mouse spermatogenic cell populations:gene expression signatures switch from meiotic-to postmeiotic-related processes at pachytene stage[J]. BMC Genomics,2016,17:294
[16]Zhou J,Wan J,Gao X,et al. Dynamic m(6)A mRNA methylation directs translational control of heat shock response[J]. Nature,2015,526(7574):591-594