The Full Length Cloning And Preliminary Functional Analysis Of A Novel Mouse Gene Biot2

Objectives:. In our previous study, a novel rat testes-specific gene, AY845219, was found from SEREX technology. To get the further information about this gene more expediently ,the full-lengthed cDNA of a novel mouse gene,Biot2, was obtained by use of homology analysis and RT-PCR technology. The features and possible functions of the novel gene were predicted by the bioinformatic analysis. Combining the results , a lot of experements were performed to study the fundamental characteristics such as the tissue expression profiling, the subcellular location and the orientation in the testis. The further work was to identify the primary functions of the gene using the RNAi technology, transfection method and constructing the mouse tumor model,etal.Methods: The full-length cDNA of the novel gene, Biot2, was obtained by RT-PCR technology and homology analysis. The expression profiling of Biot2 among the normal tisses of the sexual mature male C57 mouse and the varied tumor cells were identified by RT-PCR. The location of Biot2 in the testis and the regular expression pattern during the male sexual maturation and the spermatogenesis were studied by semiquantitative RT-PCR, Realtime RT-PCR and in situ hybridization. The subcellular localization was identified by GFP fused protein and various bioinformatic methods were used toanalyze and predict the feature and possible functions of the novel gene. Toobserve the effect on the growth of cells under the over-expression and thedown regulation of Biot2, the recombined eukaryotic expression vector andthe specific RNAi plasmid were constrcted and then transfected into theNTH3T3 and varied tumor cells. In the other hand, to make sure the results ofexperiments in vitro, the mouse tomor models were set up and therapied byinjected plasmid into the tail-vein. Morever the tumor cells transfected by thespecific RNAi plasmid after 16 hours were inoculated the BALB/c mice, andwe observed the development of tumor tissue and the lifetime of mice.Results: We cloned the full length cDNA of a novel mouse gene Biot2, whichwas differentially expressed in the testes of mouse, specially in the head ofthe mature sperm cell and adjusted regularly during the development of themale sexual maturation and the spermatogenesis. Biot2 gene was also foundin many kinds of mouse tumor cell lines, and was located in the cytoplasm byGFP fused protein and various bioinformatic methods. The growth speed ofnormal cell and the tumor cell of mouse all were inhibited (P<0.05) and thelife span of tumor bearing mice were significantly prolonged (P<0.05)compared with the control when Biot2 gene was down regulated. And thetumor of the BALB/c mice treated by shRNA plasmid developped moreslowly than that of the control(P<0.05). When the mice were killed, treatmentwith the shRNA plasmid significantly reduced mean tumor volumescompared with the controls(P<0.05).Conclusion: The full-length cDNA of the novel gene Biot2 is obtained, and the expression profiling in the normal tissues and varies tumor cells of mouse are identified. We also studied the possible relation of Biot2 gene with the testes development and the cell proliferation, which is the preliminary basis for further investigation of the novel gene's functions.