In mice, one of the major epigenetic errors associated with somatic cell nuclear transfer (SCNT) is ectopic expression of
Xist during the preimplantation period in both sexes. We found that this aberrant
Xist expression could be impeded by deletion of
Xist from the putative active X chromosome in donor cells. In male clones, it was also found that prior injection of
Xist-specific siRNA could significantly improve the postimplantation development of cloned embryos as a result of a significant repression of
Xist at the morula stage. In this study, we examined whether the same knockdown strategy could work as well in female SCNT-derived embryos. Embryos were reconstructed with cumulus cell nuclei and injected with
Xist-specific siRNA at 6–7 h after oocyte activation. RNA FISH analysis revealed that siRNA treatment successfully repressed
Xist RNA at the morula stage, as shown by the significant decrease in the number of cloud-type
Xist signals in the blastomere nuclei. However, blastomeres with different sizes (from “pinpoint” to “cloud”) and numbers of
Xist RNA signals remained within single embryos. After implantation, the dysregulated
Xist expression was normalized autonomously, as in male clones, to a state of monoallelic expression in both embryonic and extraembryonic tissues. However, at term there was no significant improvement in the survival of the siRNA-injected cloned embryos. Thus, siRNA injection was largely effective in repressing the
Xist overexpression in female cloned embryos but failed to rescue them, probably because of an inability to mimic consistent monoallelic
Xist expression in these embryos. This could only be achieved in female embryos by applying a gene knockout strategy rather than an siRNA approach.
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