RNA Interference Down-regulates The Expression Of Tctex5 Gene In Reproductive Cells Of Male Mouse

Worldwide, infertility affects approximately 10%～15% couples, in half the couples, infertility can be traced to the male. However,account for 40% of male infertility in clinic,the cause is still unknown,it offen also follow idiopathic oligospermia or azoospermi. What is even more disturbing is that the quality of human sperm is decreasing because of environmental pollutants and chemicals, which is believed to cause gene mutation. At present, Study on reproductive malfunction and Mutations of gene related to reproduction have been is a research focus in reproduction field. The Tctex5(Expression of T-complex testis expressed 5), abundantly expressed in the testis and the spermatozoa, is a novel enzyme regulating factor discovered in mouse spermatozoa. In domestic area,study on the Regulatory Mechanism of Tctex5 gene have not been reported in spermatogenesis. It is also called Ppp1r11(protein phosphatase 1 regulatory subunit 11) in human, a gene with unknown function, may involve in the sperm function control process during spermatogenesis. The Ppp1r11 encodes a specific inhibitor of protein phosphatase-1, a serine/threonine phosphatase (PP1) which roles in cells are still not clear. Possible roles of Ppp1r11 in the testis are completely unknown.RNA interference degrades mRNA specifically and potently though the mediation of corresponding double-stranded RNA and leads to post- transcriptional gene silencing. RNAi has been successfully utilized and study gene with unknown function.Double-stranded RNA (dsRNA) can trigger silencing of homologous gene expression by a mechanism termed RNAi (for RNA-mediated interference). RNAi is an evolutionarily conserved mechanism and a multistep process that involves generation of active small interfering RNA (siRNA) in vivo through the action of an RNase III endonuclease, Dicer. siRNAs have a characteristic structure,with 5'-phosphatey/ 3'-hydroxy ends and a 2-base 3'over- hang on each strand of the duplex .The resulting 21- to 23-nt siRNA mediates degradation of the complementary homologous RNA.RNAi has been used as a reverse genetic tool to study gene function in multiple model organisms, including plants, Caenorhabditis elegans, and Drosophila where large dsRNAs efficiently induce gene-specific silencing .One obstacle to achieving RNAi in mammals is that dsRNAs longer than 30 nt will activate an antiviral response, leading to the nonspecific degradation of RNA transcripts and a general shutdown of host cell protein translation . This obstacle was overcome by introducing in vitro synthesized 21 nts siRNA into mammalian cells to suppress target gene expression.In this article,The aim of this study is to design and obtain the Small interfering RNA(siRNA) which can specificly inhibit the expression of Tctex5 gene. thus, to furter study Tctex5 gene function and generate transcript-specific gene knockdown mice model provid the support of techonogy. Meanwhile ,we will can further determine and understand the potential role of Tctex5 in spermatogenesis. Though the mechanism regulating of Tctex5 gene in spermatogenesis remain to be determined.A further understanding of the molecular and celluar mechanisms of spermatogenesis may lend to the identification of navel targets for developmental biology,origin of life and breeding controI ineluding contraceptive intervention.Objective: to investigate the effect of Ppp1r11 siRNA among CRL-1715 cells , CRL-2053 cells and CRL-2196 cells.Methods: by means of siPORTTTM NeoFXTM Tranction Agent,The pre-designed Ppp1r11-specific siRNA was transfected into CRL-1715 cells,CRL-2053 cells and CRL-2196 cells, The level of messenger RNA and protein of Ppp1r11 in transfected cells was detected by RT-PCR and Western blot.Results: Donwregulation of Ppp1r11 in Ppp1r11 siRNA plasmid transfected three cells was confirmed by RT-PCR and Western blot. Compared with three cells without Ppp1r11 siRNA plasmid transfection, higher lytic activity was observed in transfected three cells.Conclusion: Down-regulating the expression of Ppp1r11 by RNAi may be suppressed significantly among CRL-1715 cells ,CRL-2053 cells and CRL-2196 cells ,thus, to furter build transgenic cell lines and transgenic RNA interference (RNAi) mouse models provid the support of techonogy and the experiment underlaying,therefor , we will preferablely study and understand the potential role of Tctex5 in male sterility.