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RNA Interference Down-regulates the Expression of Tctex5 Gene in Reproductive Cells of Male Mouse

Author: LiLei
Tutor: YangZuo；YaoYuanQing；HanYiBing
School: Fourth Military Medical University
Course: Obstetrics and Gynaecology
Keywords: RNAi siRNA Tctex5 (Ppp1r11) gene Transfection CRL-1715, CRL-2053, CRL-2196 Spermatogenesis Mouse
CLC: R711.6
Type: Master's thesis
Year: 2008
Downloads: 91
Quote: 1
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Abstract


Worldwide, human infertility affects about 10% to 15% of couples. The reason couples infertile each half [1] [2] [3]. Clinical gt; 40% of male infertility reason is unclear, and often accompanied by the presence or absence of sperm or azoospermia [4]. Even more worrying is that human sperm quality is reduced because of environmental pollution and chemical agents, and that cause gene mutations [5] [6]. The research is the the reproductive field of study one of the hot male reproductive dysfunction and reproductive-related gene mutations [7]. Tctex5 (Expression of T-complex testis expressed 5) adjustment factor gene is a novel enzyme found in the sperm of mice, abundant expression in rat testis and sperm. The Tctex5 mechanisms of gene regulation in spermatogenesis, the domestic has not been reported. The Tctex5 gene called human Ppp1r11, and regulation of a sub-unit of protein phosphatase 11 (protein phosphatase-1 inhibitor-3). The testis potential role of Ppp1r11 unclear. 1 (PP1) inhibitors: Ppp1r1 encoding a specific protein phosphorylation of serine / threonine phosphorylation. Ppp1r1 as a gene of unknown function, may be involved in the regulation of spermatogenesis process. RNAi corresponding dsRNA specifically demotion homologous mRNA in, resulting in post-transcriptional gene silencing, this technology has been successfully applied to the study of genes of unknown function. dsRNA through the RNAi mechanism mediated homologous gene silencing, RNAi is an evolutionarily conserved mechanism, a stepwise process by RNase Ⅲ-like activity of nuclease (Dicer enzyme) of the in vivo activity of small interfering RNA (siRNA ). siRNA has a characteristic structure: two monotroded end for a 5 'terminal phosphate and a 3' terminal hydroxyl group, In addition, each single-chain 3 'end were 2 to 3 highlights of the unpaired nucleotide ,21-23nt siRNA falling tone homologous RNA. RNAi has been widely used in the study of gene function in Drosophila, C. elegans, trypanosomes, mice and mammals have been found to exist RNAi phenomenon. The purpose of this study was to design and small interfering RNA specific downregulated genes expressed and detection the siRNA ability to cause the germ cells of male rats Tctex5 gene silencing. Provide technical means further to understand Ppp1r11 potential role in the process of spermatogenesis has important significance for the study of functionality of Ppp1r11 and generate the specific transcription knockout mice. Tctex5 gene regulatory mechanism is unclear. For further understanding of the molecular and cellular mechanisms of spermatogenesis, has important implications for developmental biology, the origin of life and birth control. The purpose: In Ppp1r11 gene target gene by RNA interference (RNAi) technology specifically inhibited the CRL-1715, CRL-2053, CRL-2196 cells Ppp1r11 expression. Method: for Ppp1r11 gene design for some small interfering RNA (siRNA), by chemical synthesis of Ambion transfection kit liposomes siPORTTTMNeoFXTM the siRNA transfection CRL-1715, CRL-2053, CRL-2196 three cell using RT-PCR and Western blot were used to detect the siRNA expression from the mRNA and protein levels. Results: Ppp1r11 the siRNA can significantly lowered CRL-1715, CRL-2053, CRL-2196 three cell Ppp1r11 expression levels, and with good specificity. Conclusion: CRL-1715, CRL-2053, CRL-2196 in the three cell lines using RNAi technology specific downregulation the Ppp1r11 gene expression, and lays a foundation for the establishment of transgenic cell lines, and also for the establishment of a transgenic mouse model as well as further The study Ppp1r11 spermatogenesis function provides the technical support.

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CLC: > Medicine, health > Obstetrics and Gynaecology > Gynecology > Sterility
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