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Derivation of Embryonic Germ Cells from Post Migratory Primordial Germ Cells, and Methylation Analysis of Their Imprinted Genes by Bisulfite Genomic Sequencing  

Shim, Sang Woo (Department of Bioscience and Biotechnology, Bio-Organ Research Center, Konkuk University)
Han, Dong Wook (Department of Bioscience and Biotechnology, Bio-Organ Research Center, Konkuk University)
Yang, Ji Hoon (Department of Bioscience and Biotechnology, Bio-Organ Research Center, Konkuk University)
Lee, Bo Yeon (Infertility Clinic, Department of Obstetrics and Gynecology, Kyung-Hee Medical Center)
Kim, Seung Bo (Infertility Clinic, Department of Obstetrics and Gynecology, Kyung-Hee Medical Center)
Shim, Hosup (Department of Physiology, School of Medicine, Dankook University)
Lee, Hoon Taek (Department of Bioscience and Biotechnology, Bio-Organ Research Center, Konkuk University)
Publication Information
Molecules and Cells / v.25, no.3, 2008 , pp. 358-367 More about this Journal
Abstract
The embryonic germ cell (EGCs) of mice is a kind of pluripotent stem cell that can be generated from pre- and post-migratory primordial germ cells (PGCs). Most previous studies on DNA methylation of EGCs were restricted to 12.5 days post coitum (dpc). This study was designed to establish and characterize murine EGC lines from migrated PGCs as late as 13.5 dpc and to estimate the degrees of methylation of their imprinted genes as well as of the non-imprinted locus, Oct4, using an accurate and quantitative method of measurement. We established five independent EGC lines from post migratory PGCs of 11.5-13.5 dpc from C57BL/6 ${\times}$ DBA/2 F1 hybrid mouse fetuses. All the EGCs exhibited the typical features of pluripotent cells including hypomethylation of the Oct4 regulatory region. We examined the methylation status of three imprinted genes; Igf2, Igf2r and H19 in the five EGC lines using bisulfite genomic sequencing analysis. Igf2r was almost unmethylated in all the EGC lines irrespective of the their sex and stage of isolation; Igf2 and H19 were more methylated than Igf2r, especially in male EGCs. Moreover, EGCs derived at 13.5 dpc exhibited higher levels of DNA methylation than those from earlier stages. These results suggest that in vitro derived EGCs acquire different epigenotypes from their parental in vivo migratory PGCs, and that sex-specific de novo methylation occurs in the Igf2 and H19 genes of EGCs.
Keywords
Bisulfite Sequencing; Embryonic Germ Cell(EGCs); Imprint Genes; Methylation; Mouse; Pluripotency; Primordial Germ Cell (PGCs);
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