Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Impeding Xist expression from the active X chromosome improves mouse somatic cell cloning

ABSTRACT: Cloning mammals by somatic cell nuclear transfer (SCNT) is highly inefficient because of aberrant genomic reprogramming. In addition to random reprogramming errors, we hypothesized the presence of specific errors as evidenced by common anomalies among clones. We found that Xist, which normally inactivates one of the two X chromosomes in females, was ectopically expressed from the active X (Xa) chromosome in cloned mouse embryos of both sexes. Deletion of Xist on Xa normalized global gene expression and produced about a 10-fold increase in cloning efficiency. We also identified an Xist-independent mechanism that specifically downregulated a subset of X-linked genes through somatic-type repressive histone blocks. Thus, we have identified nonrandom reprogramming errors in mouse cloning, which provide promising targets for breakthroughs in SCNT cloning technology. Gene expression were measured in mouse in vitro fertilized and somatic cell cloned blastocysts. More than three biological replicates were performed in each group using defferent nuclear donor cells.

ORGANISM(S): Mus musculus

SUBMITTER: Kimiko Inoue

PROVIDER: E-GEOD-23181 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Impeding Xist expression from the active X chromosome improves mouse somatic cell nuclear transfer.

Inoue Kimiko K Kohda Takashi T Sugimoto Michihiko M Sado Takashi T Ogonuki Narumi N Matoba Shogo S Shiura Hirosuke H Ikeda Rieko R Mochida Keiji K Fujii Takashi T Sawai Ken K Otte Arie P AP Tian X Cindy XC Yang Xiangzhong X Ishino Fumitoshi F Abe Kuniya K Ogura Atsuo A

Science (New York, N.Y.) 20100916 6003

Cloning mammals by means of somatic cell nuclear transfer (SCNT) is highly inefficient because of erroneous reprogramming of the donor genome. Reprogramming errors appear to arise randomly, but the nature of nonrandom, SCNT-specific errors remains elusive. We found that Xist, a noncoding RNA that inactivates one of the two X chromosomes in females, was ectopically expressed from the active X (Xa) chromosome in cloned mouse embryos of both sexes. Deletion of Xist on Xa showed normal global gene e ...[more]

PMID: 20847234

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Project description:The great majority of embryos generated by somatic cell nuclear transfer (SCNT) display defined abnormal phenotypes after implantation, such as an increased likelihood of death and abnormal placentation. To gain better insight into the underlying mechanisms, we analyzed genome-wide gene expression profiles of day 6.5 postimplantation mouse embryos cloned from three different cell types (cumulus cells, neonatal Sertoli cells and fibroblasts). The embryos retrieved from the uteri were separated into embryonic (epiblast) and extraembryonic (extraembryonic ectoderm and ectoplacental cone) tissues and were subjected to gene microarray analysis. Genotype- and sex-matched embryos produced by in vitro fertilization (IVF) were used as controls. Principal component analysis revealed that whereas the gene expression patterns in the embryonic tissues varied according to the donor cell type, those in extraembryonic tissues were relatively consistent across all groups. Within each group, the embryonic tissues had more differentially expressed genes (DEGs) (> 2-fold vs. controls) than did the extraembryonic tissues (P < 1.0 M-CM-^W 10M-bM-^@M-^S26). In the embryonic tissues, one of the common abnormalities was upregulation of Dlk1, a paternally imprinted gene. This might be a potential cause of the occasional placenta-only conceptuses seen in SCNT-generated mouse embryos (1M-bM-^@M-^S5% per embryo transferred in our laboratory), because dysregulation of the same gene is known to cause developmental failure of embryos derived from induced pluripotent stem cells. There were also some DEGs in the extraembryonic tissues, which might explain the poor development of SCNT-derived placentas at early stages, although these alterations were not always statistically significant for all three SCNT groups because of variability. These findings suggest that SCNT affects the embryonic and extraembryonic development differentially and might cause further deterioration in the embryonic lineage in a donor cell-specific manner. This could explain donor cell-dependent variations in cloning efficiency using SCNT. Comparative gene expression analyses using E4.0 cloned blastocysts were performed by microarray. Cloned embryos were produced with cumulus cells by two different methods (cell fusion and nuclear injection). As controls, sex- and genotype-matched embryos produced by in vitro fertilization were used.

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Impeding Xist expression from the active X chromosome improves mouse somatic cell cloning

Publications

Impeding Xist expression from the active X chromosome improves mouse somatic cell nuclear transfer.

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