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

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Author(s)

Abstract

In mice, one of the major epigenetic errors associated with somatic cell nuclear transfer (SCNT) is ectopic expression of <i>Xist</i> during the preimplantation period in both sexes. We found that this aberrant <i>Xist</i> expression could be impeded by deletion of <i>Xist</i> from the putative active X chromosome in donor cells. In male clones, it was also found that prior injection of <i>Xist</i>-specific siRNA could significantly improve the postimplantation development of cloned embryos as a result of a significant repression of <i>Xist</i> 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 <i>Xist</i>-specific siRNA at 6–7 h after oocyte activation. RNA FISH analysis revealed that siRNA treatment successfully repressed <i>Xist</i> RNA at the morula stage, as shown by the significant decrease in the number of cloud-type <i>Xist</i> signals in the blastomere nuclei. However, blastomeres with different sizes (from “pinpoint” to “cloud”) and numbers of <i>Xist</i> RNA signals remained within single embryos. After implantation, the dysregulated <i>Xist</i> 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 <i>Xist</i> overexpression in female cloned embryos but failed to rescue them, probably because of an inability to mimic consistent monoallelic <i>Xist</i> expression in these embryos. This could only be achieved in female embryos by applying a gene knockout strategy rather than an siRNA approach.

Journal

  • Journal of Reproduction and Development

    Journal of Reproduction and Development 59(3), 231-237, 2013-06-01

    THE SOCIETY FOR REPRODUCTION AND DEVELOPMENT

References:  25

Codes

  • NII Article ID (NAID)
    10031184751
  • NII NACSIS-CAT ID (NCID)
    AA10936678
  • Text Lang
    ENG
  • Article Type
    ART
  • ISSN
    09168818
  • NDL Article ID
    024616784
  • NDL Call No.
    Z54-H305
  • Data Source
    CJP  NDL  J-STAGE 
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