RNAi-mediated Knockdown of Xist Does Not Rescue the Impaired Development of Female Cloned Mouse Embryos

In mice, one of the major epigenetic errors associated with somatic cell nucleartransfer (SCNT) is ectopic expression of Xist during the preimplantationperiod in both sexes. We found that this aberrant Xist expression couldbe impeded by deletion of Xist from the putative active X chromosome indonor cells. In male clones, it was also found that prior injection ofXist-specific siRNA could significantly improve the postimplantationdevelopment of cloned embryos as a result of a significant repression ofXist at the morula stage. In this study, we examined whether the sameknockdown strategy could work as well in female SCNT-derived embryos. Embryos werereconstructed with cumulus cell nuclei and injected with Xist-specificsiRNA at 6–7 h after oocyte activation. RNA FISH analysis revealed that siRNA treatmentsuccessfully repressed Xist RNA at the morula stage, as shown by thesignificant decrease in the number of cloud-type Xist signals in theblastomere nuclei. However, blastomeres with different sizes (from “pinpoint” to “cloud”)and numbers of Xist RNA signals remained within single embryos. Afterimplantation, the dysregulated Xist expression was normalizedautonomously, as in male clones, to a state of monoallelic expression in both embryonicand extraembryonic tissues. However, at term there was no significant improvement in thesurvival of the siRNA-injected cloned embryos. Thus, siRNA injection was largely effectivein repressing the Xist overexpression in female cloned embryos but failedto rescue them, probably because of an inability to mimic consistent monoallelicXist expression in these embryos. This could only be achieved in femaleembryos by applying a gene knockout strategy rather than an siRNA approach.