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 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.
RIKEN BioResource Center, Ibaraki 305-0074, Japan.
Mami
M
Oikawa
Shogo
S
Matoba
Kimiko
K
Inoue
Satoshi
S
Kamimura
Michiko
M
Hirose
Narumi
N
Ogonuki
Hirosuke
H
Shiura
Michihiko
M
Sugimoto
Kuniya
K
Abe
Fumitoshi
F
Ishino
Atsuo
A
Ogura
D000483:Alleles; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D019976:Cloning, Organism / Q000379:methods*; D054885:Cumulus Cells / Q000378:metabolism; D044127:Epigenesis, Genetic; D005260:Female; D018507:Gene Expression Regulation, Developmental; D055785:Gene Knockdown Techniques; D006877:Hydroxamic Acids / Q000737:chemistry; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D009028:Morula / Q000378:metabolism; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000166:cytology; D034622:RNA Interference; D062085:RNA, Long Noncoding / Q000235:genetics* / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism
D006877:Hydroxamic Acids; D062085:RNA, Long Noncoding; D034741:RNA, Small Interfering; C077885:XIST non-coding RNA; C012589:trichostatin A