Effect Of Xist Gene Repression On Early Development Of Bovine Scnt Embryos

Xist is an X-linked gene that produces a noncoding RNA, and it is one of the first imprinted genes to be expressed in the early embryo with expression beginning at zygotic genome activation. Although Xist is known to be involved in X-chromosome inactivation in females, the function of Xist RNA is not fully understood. Many genes are aberrantly expressed in cloned embryos, and Xist is among these genes. This experiment investigates feasibility of TALER vectors transfected into of fetal bovine fibroblast to control genetic expression of Xist. and also researches impact of Xist gene control on early development of cloned bovine embryo by making cloned embryo with cells that suppress Xist expression as the donor. The results showed this experiment applies specificity Xist TALER vector, which is transfected into fibroblast of female fetal bovine fibroblasts, and mCherry is used as reporter gene of vector transfection efficiency. Flow cytometry is used to select positive cells and extract RNA directly at first; cDNA is amplified with inverse transcription, and then relative expression level detection of Real time RT-PCR. Xist between it and the isogenic cells. According to the result, relative expression level of the treatment group has been reduced by93.85%compared with that of control group, indicating the vector transfection system designed in this experiment can effectively suppress genetic expression of Xist. Transfection cells with positive Xist expression suppression are selected for SCNT experiment, and untreated cell is used for comparison at the same time. Finally, cleavage rate,8-cell development rate, morula development rate and blastaea development rate of treatment group vs. control group are78.8%vs75.1%(P>0.05, no obvious difference),54.4%vs.50.6%(P>0.05, no obvious difference),12.3%vs.27.8%(P<0.01, significant difference) and0vs.26.6%(P<0.01, significant difference). According to the above results, These results show that TALER-mCherry vectors specific repress Xist gene expression in female fetal bovine fibroblast. Repression of Xist in donor cells promotes the2-8cell of cloned embryo is found slightly higher than that in control group, while obvious suppression effect is shown to cloned embryo development at blastula stage. However, development of morula/blastocyst decreased significantly. Therefore, its mechanism needs further investigation.