The Establishment And Phenotypic Analysis Of Transgenic Mice Model With Speedy A Controllable Expression

Objective Mammalian spermatogenesis is a highly complex process of cell division and differentiation, many specific spermatogenic cell gene’s expression and regulation has the developmental stage-specific features, and any exception may cause infertility. Therefore, research the testis-specific genes which play the important roles in spermatogenesis, and explore the expression profile of these genes and their biological functions in spermatogenesis in-depth will contribute to understanding the internal mechanism of spermatogenesis, preventing sperm abnormalities caused by the occurrence of male infertility, learning male sexual function decline, and developing male contraceptive drug and health medicine. Speedy/Ringo genes are a class of cyclin-dependent kinase (CDK) activator which were newly discovered and play important roles in spermatogenesis. Tetracycline (tetracycline, Tet) regulation of gene expression systems can achieve controllable expression in time and space and provide effective solutions for the study of gene function. This paper aims to establish transgenic mice model with Speedy A-RNAi controllable expression based on Tet-On system, to investigate the application of Tet-On system in spermatogenesis related gene’s research and explore the effects on spermatogenesis with Speedy A knockdown.Methods According to RNA interference sequence design principle designed RNA interference target sequence, in vitro experiment was used to screen the effective interference fragment of Speedy A, and constructed the vector (pRP.EX2d-TRE>shSpdya) which contains tetracycline-responsive element and the interference fragment; The transgenic mice model with Speedy A-RNAi were obtained by microinjected the vector called pRP.EX2d-TRE>shSpdya which was established by Gateway technology; the homogeneous mice were selected and mated with reverse tet-controlled transactivator transgenic mice which regulated by Tetracycline; feed the mice with doxycycline (Dox) induced the down-regulated expression of Speedy A by induced the expression of RNA interference fragment; the differential expression between model group and control group were detected by real-time fluorescent quantitative PCR and Western Blot; observe the morphological changes in testis by HE, and apoptosis in testicular tissue of mice were detected by TUNNEL.Results The experiment designed 4 RNA interference fragment to knockdown the expression of Speedy A gene, the most effective one was screened in vitro; Real-time PCR detected the most effective interference fragment of Speedy A knockdown the gene’s expression more than 75%; the experiment successfully constructed PDOWN-shSpdya entry clone and pRP.EX2d-TRE> shSpdya expression clone; establish transgenic mice model with Speedy A-RNAi controllable expression based on Tet-On system, compared with the control group, the levels of Speedy A of model group remarkably decreased and found a large number of spermatogenic cell apoptosis occurred in mouse testis of model group.Conclusions The transgenic mice model with Speedy A-RNAi controllable expression based on Tet-On system had been established, this means the study of spermatogenesis related gene’s biological function research in vivo based on Tet-On system was feasible. Preliminary analysis found that the down-regulated expression of Speedy A causes abnormal spermatogenic cells apoptosis in testis. The success of the model’establish in this paper laid the foundation for further analysis of the function of Speedy A in the male reproductive system and its mechanism of action.