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

Access this Article

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
  • DOI
    10.1262/jrd.2012-195
  • Data Source
    CJP  NDL  J-STAGE 
Page Top