RNAi-mediated knockdown of Xist improves development of the female buffalo (Bubalus bubalis) nuclear transfer embryos

Xist plays a critical role in the X-chromosome inactivation (XCI), an important epigenetic reprogramming of somatic cell nuclear transfer (SCNT) embryos. Modulation of Xist expression enhanced the developmental ability of mouse cloned embryos. However, the roles of Xist in buffalo SCNT embryos remain unknown. In this study, we investigated the methylation and expression status of Xist in different genders of buffalo donor cells and various stages (two-cell, eight-cell, morula and blastocyst) of in vitro fertilization (IVF) and SCNT embryos. The methylation of Xist in SCNT-♀ and SCNT-♂ embryos was aberrant hypomethylation compared with the buffalo foetal fibroblast (♀-BFF and ♂-BFF), IVF-♀ and IVF-♂ embryos. At the eight-cell stage, Xist expression was significantly higher in SCNT-♀ embryos compared with those in SCNT-♂, IVF-♀ and IVF-♂ embryos (P < 0.05). Meanwhile, no significant difference was found between IVF-♀ and IVF-♂ embryos (P > 0.05). Accordingly, we suppressed Xist expression by RNAi-Xist in SCNT-♀ embryos. Results showed that injection of Xist-shRNA significantly improved the morula and blastocyst rates (P < 0.05). These results indicated that correcting the abnormal expression of the Xist gene contributed to the development of SCNT-♀ embryos.