Nuclear Transfer Alters the DNA Methylation Status of Specific Genes in Fertilized And Parthenogenetically Activated Mouse Embryonic Stem Cells

¹ Laboratory for Genomic Reprogramming, Center for Developmental Biology, RIKEN Kobe institute, 2-2-3 Minatojima-minamimachi, Kobe 650-0047, Japan
² Department of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo 101-0062, Japan

^* To whom correspondence should be addressed. E-mail: hikichi{at}cdb.riken.jp.

	Abstract

Therapeutic cloning has been demonstrated successfully using nuclear transfer (NT) techniques to generate embryonic stem cells (NT-ES cells). Mice can be cloned from adult somatic cells or ES cells by NT and such cloned embryos can be used to establish new NT-ES cell lines. However, ES cells derived from parthenogenetic embryos (pES cells) show epigenetic disorders and low potential for normal differentiation unless used to produce subsequent generations of NT-ES lines. Thus, enucleated oocytes can initialize epigenetic modification, but the extent and efficacy of this remains unclear. In this study we aimed to clarify why the contribution rate of pES cells appears to improve after NT. We compared gene expression profiles between pES and NT-pES cell lines using DNA microarray analysis and allele-specific DNA methylation analysis. Although changes in expression level were observed for 4% of 34,967 genes, only 81 (0.2%) showed common changes across multiple cell lines. In particular, the expression level of a paternally expressed gene, U2af1-rs1, was increased significantly in all NT-pES cell lines investigated. The methylation status at the upstream differentially methylated region (DMR) of U2af1-rs1 was also changed significantly after NT. This was observed in NT-pES cells, but also in conventionally produced NT-ES cells, which has never been reported previously. These results suggest that NT affects the epigenetic status of a few gene regions in common and that a change in the methylation status U2af1-rs1 could be used as a genetic marker to investigate the effects of NT.