The Construction Of RNAi Viral Vector And Observation Of Sex Control From Mus Zyf Gene

In this study, the targeting protocols of retroviral vector-mediated RNAi are used to inhibit the expression of the related gender determination genes Zfy, in order to study the associative function of Zfy gene in sprematogenesis and the change of mice offspring sex ratio.Constructing the retroviral vectors (Retrovirus/Zfy-shRNA) and plasmid vectors (Psilencer/Zfy-shRNA), and using them transfects the sperm cells in mice, then we can use the qRT-PCR to detect the expression level of the mRNA in each group Zfy gene, confirmation of silence effect by interference vector for Zfy gene. Large quantity of virus particles were packed and plasmid vector were prepared and injected into testis of male rat. Every7d injection, continuous injection of4times, testicular tissues of some male mice was collected at14d after the last injection and detected the expression level of Zfy gene with real time PCR(qRT-PCR); the other male mice were mated with superovulated females as1:1, three groups mice were mated at5days,and observed the sex ratio.The results showed that Retrovirus/Zfy-shRNA and plasmid vector Psilencer/Zfy-shRNA were transfected into mouse sperm cells, the expression level of Zfy gene mRNA decreased significantly (P<0.01)than empty vector and blank control group; after testicular injection of Retrovirus/Zfy-shRNA and plasmid vector Psilencer/Zfy-shRNA,the result showed that the expression level of Zfy gene mRNA decreased significantly (P<0.01) than empty vector and saline group. However,both controls had not significant difference (P>0.05); the male mice, which was injected the retroviral vector in testis, were mated every5days,and got71.43%of the average mice offspring rate were female, which was significantly higher than the empty vector control group (P<0.01), higher than the saline control group (P<0.05). Compared with the control group, the average rate of female offspring of the mated was gradually reduced at2ed,3rd, the difference was not significant (P>0.05) Simultanceously, the male group of testicular injection with plasmid vectors Psilencer/Zfy-shRNA were mated every5d, getting the1st and2ed mating average rate of female offspring were71.00%and73.21%respectively, which was significantly higher than the empty vector control group (P<0.01), higher than the saline control group (P<0.05);3rd the average female mating offspring was63.64%, higher than the empty vector control group (P<0.05), but similar to the saline control group (P>0.05).Finally,we dram the following conclusions, constructed RNAi retrovirus vector successfully inhibits the expression of mice Zfy gene. Control mice gender before fertilization through the interference Zfy gene expression, at the same time, also laid the foundation for the study of other animals before fertilization gender control.