RNAi-mediated knockdown of Xist does not rescue the impaired development of female cloned mouse embryos

Abstract

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.

Fig. 1.

RNA-FISH analyses of Xist in cumulus cell-derived cloned embryos (A and B), female IVF embryos (C), and female ES cell-derived embryos (D). The right panels show the ratios of blastomeres classified according to the number of cloud or pinpoint signals of Xist RNA. A: At the morula stage (72 h after activation), the Xist siRNA treatment significantly increased the proportion of blastomeres without cloud signals (counting those with two pinpoints + one pinpoint + no signal) (19 vs. 76% on average; P<0.005). B: At the blastocyst stage (96 h after activation), there was no significant difference in the proportion of blastomeres without cloud signals between the control and Xist-specific siRNA groups (40 vs. 41%; P>0.05). C: In normally fertilized female embryos, the majority of blastomeres showed expression of one cloud signal in controls and one pinpoint signal in Xist-specific siRNA-injected embryos. D: In embryos cloned from female ES cells, the expression patterns or Xist with or without Xist-specific siRNA treatment were similar to those found in cumulus cell-derived cloned embryos (see A). (Scale bar, 50 μm).

Fig. 2.

A: QPCR for Xist in cumulus cell-derived cloned embryos injected with Xist-specific siRNA and cultured for 72 h (morula) and 96 h (blastocyst). There were no differences among the three experimental groups, including IVF-derived embryos, at either the morula or blastocyst stages (P>0.05). B: The XCI status of cloned embryos at embryo day 5.5. A representative image of RNA FISH for Xist (red) with nuclear staining (blue) in cumulus cell-derived cloned embryos (left), and the ratio of cells classified according to the number of Xist RNA signals (right). Most cells in the epiblast (Epi) and extraembryonic ectoderm (EXE) showed a single Xist signal in the nucleus. (Scale bar, 50 μm).