The Effect Of RNAi And CRISPR/Cas9-based Y Chromosome Gene Targeting On Male Mouse Embryonic Development

Sex control technology has important application value in animal husbandry.Most of the genes encoding genes in mammalian Y are related to sexual development. By regulating the expression or function of genes on Y chromosome, the sex ratio may be changed.This study selected the Dby gene on the Y chromosome as the target gene, the gene mRNA in sperm maturation and early embryonic development were able to be detected,implies that the function of the gene may with the male embryonic development. We used the method of RNA interference to study the effect of down-regulation of the expression of Dby gene in mouse early embryos on the development of male embryo and the reproductive ability of male mice after birth. Dby mRNA coding region of the search for the four target sites, lentiviral vectors expressing sh RNA were constructed and transfected into MC3T3-E1 cell lines screened has showed the best inhibition effect. It can make the cell Dby gene expression was down regulated significantly to the control group of 14%(P<0.05).According to the shrna1 synthetic si RNA design and of mouse fertilized eggs of male pronucleus microinjection and found si RNA to cultured in vitro to the eight cell stage embryos Dby gene expression significantly dropped to control the 6.1%(P<0.01); to transplant to receptors in mouse embryonic development and sex ratio did not affect, but the breeding male mice after conception rate had significant effect(P<0.05), also had a certain inhibitory effect on the testis of male mice and development. Suggesting that in the early mouse embryo using RNA interference technology can effectively inhibit the Dby gene expression, but Dby gene expression was downregulated in male embryonic development did not affect, on postnatal male mouse reproductive ability to cause certain effect.At the same time, this study selected the multi copy gene Rbmy on the Y chromosomeas the target gene, using the CRISPR/Cas9 system to specifically cut the Rbmy gene, and study the possibility of the CRISPR/Cas9 system to destroy the Y chromosome. We found 6target sites for Rbmy gene, and constructed the corresponding eukaryotic expression vector,prokaryotic expression vector and target vector. We transfected 293 T cell lines with Cas9,sg RNA and fluorescence report. By detecting the fluorescence efficiency and fluorescence intensity, we can predict the cutting efficiency of CRISPR/Cas9 system at different target sites. The results showed that sg RNA6 had the best cutting effect on 293 T cell lines, and the fluorescence efficiency was 52.64%, which was significantly higher than that of the control group, which was close to the positive control. We then for the in vitro transcription of sg RNA, using sg RNA in vitro can forms the characteristics of endonuclease with cas9 protein and in vitro detection the CRISPR/Cas9 system of target sequence cutting efficiency,found sg RNA6 on the target sequence cutting efficiency is almost 100%, and cell level consistent with the results that sg RNA6 can be used for next step of Y chromosome in male embryos were cutting of the study.