Effects of RNAi-mediated knockdown of Xist on the developmental efficiency of cloned male porcine embryos

Xist is an X-linked gene responsible for cis induction of X chromosome inactivation. Studies have indicated that Xist is abnormally activated in the active X chromosome in cloned mouse embryos due to loss of the maternal Xist-repressing imprint following enucleation during somatic cell nuclear transfer (SCNT). Inhibition of Xist expression by injecting small interfering RNA (siRNA) has been shown to enhance the in vivo developmental efficiency of cloned male mouse embryos by more than 10-fold. The purpose of this study was to investigate whether a similar procedure can be applied to improve the cloning efficiency in pigs. We first found that Xist mRNA levels at the morula stage were aberrantly higher in pig SCNT embryos than in in vivo fertilization-derived pig embryos. Injection of a preselected effective anti-Xist siRNA into 1-cell-stage male pig SCNT embryos resulted in significant inhibition of Xist expression through the 16-cell stage. This siRNA-mediated inhibition of Xist significantly increased the total cell number per cloned blastocyst and significantly improved the birth rate of cloned healthy piglets. The present study contributes useful information on the action of Xist in the development of pig SCNT embryos and proposes a new method for enhancing the efficiency of pig cloning.