关键词: RNAi, Fankl, 293T细胞

Abstract: Objective Construct and screening RNAi vector of mouse testis tissuespecific gene Fankl, to lay a foundation of further study cn gene function of Fankl and gene therapy of male infertility related to Fankl. Methods Designing 2 interference sequence using http://www.dharmacon.com/ online soft according to Fankl cDNA sequence，and clone them to the plasmid vector pSuper-neo-GFP with HI promoter, amplifying Fankl complete sequence from adult mouse testis, and then construct red fluorescent protein fusion vector incorporating Fankl, pdsRED-Fankl. Cotransfecting pdsRED-Fankl with two interference vectors and control to 293T cells respectively using liposome method, to observe the inhibitory effect of siRNA to Fankl and the expression of red fluorescent protein, and to detect the expression of FANK1 mRNA and protein by RT-PCR and Western blot. Results PCR analysis confirmed that recombinant plasmids pSuper-shFankl 631#, pSuper-shFankl 247# and pdsRED-Fankl have been successfully constructed. Sequencing analysis were completely correct. Under fluorescent microscope, shFankl 631、247 significantly inhibit red fluorescent protein expression 36h after tansfection 293Tcell. The inhibitory rates were 95% and 90% respectively. RT-PCR and Western blot showed that Fankl gene was inhibited both on mRNA and protein level (P＜0.05). Conclusions The RNAi vector of mouse testis tissuespecific gene Fankl were constructed successfully, lay a foundation for establishing Fankl knock-down mouse and studying the role of Fankl in spermatogenesis.

Key words: RNAi, Fankl, 293T cell