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Rnai-Mediated Knockdown of Xist Can Rescue the Impaired Postimplantation Development of Cloned Mouse Embryos

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Rnai-Mediated Knockdown of Xist Can Rescue the Impaired Postimplantation Development of Cloned Mouse Embryos RNAi-mediated knockdown of Xist can rescue the impaired postimplantation development of cloned mouse embryos Shogo Matobaa,1, Kimiko Inouea,b,1,2, Takashi Kohdac,1, Michihiko Sugimotoa,1, Eiji Mizutania, Narumi Ogonukia, Toshinobu Nakamurad,3, Kuniya Abea, Toru Nakanod, Fumitoshi Ishinoc,2, and Atsuo Oguraa,b,e,2 aRIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan; bGraduate School of Life and Environmental Science, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan; cDepartment of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510, Japan; dGraduate School of Frontier Bioscience, Osaka University, Suita, Osaka 565-0871, Japan; and eCenter for Disease Biology and Integrative Medicine, Faculty of Medicine, University of Tokyo, Tokyo 113-0033, Japan Edited by R. Michael Roberts, University of Missouri, Columbia, MO, and approved October 12, 2011 (received for review August 5, 2011) Cloning mammals by somatic cell nuclear transfer (SCNT) is highly vived to term, resulting in an eight- to ninefold increase in overall inefficient. Most SCNT-generated embryos die after implantation cloning efficiency (6). These findings indicate that many, if not because of unidentified, complex epigenetic errors in the process of all, of the mechanisms that compromise the development of postimplantation embryonic development. Here we identify the clones can be attributed to ectopic expression of the Xist gene in most upstream level of dysfunction leading to impaired development mice. The XIST gene is also known to be aberrantly expressed in of clones by using RNAi against Xist, a gene responsible for X chro- bovine and pig SCNT-derived embryos and is implicated in their mosome inactivation (XCI). A prior injection of Xist-specific siRNA into prenatal death (7–9). Thus, correction of X-linked gene ex- reconstructed oocytes efficiently corrected SCNT-specific aberrant pression is one of the most promising strategies now conceivable Xist expression at the morula stage, but failed to do so thereafter for improving the efficiency of mammalian SCNT. fi Xist at the blastocyst stage. However, we found that shortly after implan- Despite its potential ef cacy, the -knockout approach is im- fi tation, this aberrant XCI status in cloned embryos had been corrected practical because it causes irreversible modi cation of the donor BIOLOGY genome. Furthermore, gene targeting is still difficult for animal autonomously in both embryonic and extraembryonic tissues, prob- DEVELOPMENTAL fi ably through a newly established XCI control for postimplantation species other than mouse because of the dif culty in establishment embryos. Embryo transfer experiments revealed that siRNA-treated of germ line-competent embryonic stem cells and the low cost-ef- fectiveness of designed nucleases (10). In addition, it is still unclear embryos showed 10 times higher survival than controls as early as Xis embryonic day 5.5 and this high survival persisted until term, result- from knockout studies how and when the ectopic expression of t ing in a remarkable improvement in cloning efficiency (12% vs. 1% in affects the development of cloned embryos. The most probable controls). Importantly, unlike control clones, these Xist-siRNA clones solution to overcome these problems might be RNA interference (RNAi)-mediated gene knockdown, which works transiently and at birth showed only a limited dysregulation of their gene expression, – indicating that correction of Xist expression in preimplantation em- does not alter genomic DNA sequences (11 13). This strategy is easily applicable to many mammalian species and could provide us bryos had a long-term effect on their postnatal normality. Thus, con- with clues to understanding the critical period of gene expression trary to the general assumption, our results suggest that the fate of that might allow cloned embryos to survive to term. In this study, we cloned embryos is determined almost exclusively before implantation examined whether RNAi-mediated knockdown against Xist could by their XCI status. Furthermore, our strategy provides a promising ameliorate the poor development of cloned mouse embryos by breakthrough for mammalian SCNT cloning, because RNAi treatment injecting a short interfering RNA (siRNA) into reconstructed of oocytes is readily applicable to most mammal species. embryos. As exact quantitative adjustment by conventional RNAi is technically difficult, we primarily focused on the silencing of Xist somatic cell cloning | RNA FISH | DNA micro array | Tsix | trichostatin A RNA in male cloned embryos with only a single X chromosome. omatic cell nuclear transfer (SCNT) is a unique technology Results Sfor endowing the somatic cell genome with totipotency by Injected Xist-siRNA Repressed Ectopic Xist Expression at the Morula genomic reprogramming (1). This technique has a distinct ad- Stage but Not Later in Cloned Embryos. First, we examined the vantage over other similar biotechnologies, such as generating validity of an siRNA construct using parthenogenetic embryos, induced pluripotent stem cells, because a single somatic cell can which express the Xist gene from the morula stage onward (14). give rise to a new individual with exactly the same genome as that of the donor cell (2, 3). Therefore, SCNT could become an in- valuable tool with a broad range of applications including bi- Author contributions: S.M., K.I., and A.O. designed research; S.M., K.I., T.K., M.S., E.M., ological drug manufacturing, regenerative medicine, endangered N.O., and A.O. performed research; T. Nakamura contributed new reagents/analytic tools; species preservation, and commercial animal breeding. However, T.K., K.A., T. Nakano, and F.I. analyzed data; and A.O. wrote the paper. the success rate of cloning mammals by SCNT is low, pre- The authors declare no conflict of interest. dominantly because of the developmental arrest of cloned em- This article is a PNAS Direct Submission. bryos after embryo transfer (1, 4). Freely available online through the PNAS open access option. Our recent in-depth gene expression analysis of cloned mouse Data deposition: The microarray data reported in this paper have been deposited in the blastocysts revealed ectopic expression of the noncoding RNA Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. Xist—responsible for X chromosome inactivation (XCI) in fe- GSE33208). — male cells (5) from the active X chromosome in both male and 1S.M., K.I., T.K., and M.S. contributed equally to this work. Xist female clones (6). This ectopic expression of had a global 2To whom correspondence may be addressed. E-mail: [email protected], fishino.epgn@ adverse effect on the gene expression of cloned embryos, be- mri.tmd.ac.jp, or [email protected]. Xist cause normalization of expression by genetic knockout re- 3Present address: Department of Animal Bio-Science, Nagahama Institute of Bio-Science markably improved the expression patterns of not only X-linked and Technology, Nagahama, Shiga 526-0829, Japan. genes but also a number of autosomal genes in the cloned em- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. bryos. As a result, many of the knockout-cloned embryos sur- 1073/pnas.1112664108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1112664108 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 We found that the siRNA treatment effectively decreased the and C). We also investigated the XCI status of cloned blastocysts by Xist RNA level in the resulting blastocysts when injected into staining for trimethylated histone H3 at lysine 27 (H3K27me3), oocytes at 6 h postactivation (Fig. S1). As parthenogenetic em- a marker for repressed chromatin state in the inactive X chromo- bryos arrest their development at midgestation, we next injected some (15). In blastocysts of both Xist-siRNA–treated and control the Xist-siRNA into in vitro fertilization (IVF)-derived embryos embryos, the majority (>65%) of inner cell mass cells and tro- to determine whether the knockdown of Xist would affect normal phectoderm cells were positive for punctate H3K27me3 staining postimplantation development. After embryo transfer, 44% of (Fig. 1D), confirming the findings by RNA-FISH analysis (Fig. 1B). the siRNA-injected embryos reached term, showing a nearly 1:1 These results collectively indicate that the injected Xist-siRNA was sex ratio, with no significant difference from that of control effective up to about 72 h of embryo development but that the embryos (P > 0.05 by Fisher’s exact test) (Table S1). This result efficacy diminished thereafter. indicates that Xist-siRNA did not affect the normal development of embryos of either sex. Therefore, in the next series of experiments, Injected Xist-siRNA Normalized Global Gene Expression of Cloned the same regimen was used for male SCNT embryos reconstructed Embryos at the Morula Stage. We then examined the effect of using nuclei from neonatal Sertoli cells. Quantitative reverse Xist-siRNA injection on the gene expression pattern of cloned transcription–PCR (RT-PCR) analyses revealed that Xist tran- embryos by DNA microarray analysis using single embryos (6). scripts were first detected at the four-cell stage in cloned embryos When relative expression levels of control cloned morulae were and reached a steady level at the morula and blastocyst stages (Fig. plotted on the positions of the X chromosome, X-linked genes were 1A). When cloned embryos were pretreated with Xist-siRNA largely down-regulated over the entire chromosome (Fig. 2A)be- (hitherto termed Xist-siRNA embryos), the Xist level was reduced cause of the high ectopic Xist expression at this stage, as shown in significantly compared with control siRNA embryos at the morula Fig. 1. By contrast, in cloned Xist-siRNA embryos, the relative ex- stage but not at the blastocyst stage (Fig. 1A). The effect of RNAi pression levels were elevated considerably in a chromosome-wide on ectopic Xist expression was further examined with Xist RNA manner, indicating that the repression of ectopic Xist had a pro- fluorescent in situ hybridization (RNA FISH).
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