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Gabbine Wee,Yongsub Kim,Hyojin Kim,Choong-il Lee,Jin-Soo Kim 한국당과학회 2012 한국당과학회 학술대회 Vol.2012 No.1
Fukutin is a putative glycosyltransferase protein that is involved in the glycosylation of α-dystroglycan, and highly expressed in skeletal muscle, heart and brain. Therefore, the protein could play an important role in muscle and brain development. Mutations in this gene have been reported as a cause of Fukuyama congenital muscular dystrophy (FCMD), Walker-Warburg syndrome (WWS), limb-girdle muscular dystrophy type 2M (LGMD2M), and dilated cardiomyopathy type 1X (CMD1X). However, the biological functions of this protein and molecular mechanisms of these genetic diseases remain elusive. To serve disease models for functional study, we disrupted the fukutin gene using a transcription activator like effector nuclease (TALEN) that is an artificial restriction enzyme composed of DNA binding domain and Fok I nuclease. TALENs induce site-specific DNA double-strand breaks (DSBs) in the genome, whose repair via error-pron non-homologous end-joining gives rise to targeted mutations. We used a surrogate reporter to enrich cells that contain TALEN-induced mutations in HeLa cells and isolated gene knockout clones. We propose that engineered TALENs are powerful tools for functional studies of glycosylation in cell lines.
Wee, Gabbine,Shim, Jung-Jae,Koo, Deog-Bon,Chae, Jung-Il,Lee, Kyung-Kwang,Han, Yong-Mahn Journals of Reproduction and Fertility 2007 Reproduction Vol.134 No.6
<P>Epigenetic reprogramming is a prerequisite process during mammalian development that is aberrant in cloned embryos. However, mechanisms that evolve abnormal epigenetic reprogramming during preimplantation development are unclear. To trace the molecular event of an epigenetic mark such as DNA methylation, bovine fibroblasts were epigeneticallyaltered by treatment with trichostatin A (TSA) and then individually transferred into enucleated bovine oocytes. In the TSA-treated cells, expression levels of histone deacetylases and DNA methyltransferases were reduced, but the expression level of histone acetyltransferases such as Tip60 and histone acetyltransferase 1 (HAT1) did not change compared with normal cells. DNA methylation levels of non-treated (normal) and TSA-treated cells were 64.0 and 48.9% in the satellite I sequence (P < 0.05) respectively, and 71.6 and 61.9% in the alpha-satellite sequence respectively. DNA methylation levels of nuclear transfer (NT) and TSA-NT blastocysts in the satellite I sequence were 67.2 and 42.2% (P < 0.05) respectively, which was approximately similar to those of normal and TSA-treated cells. In the alpha-satellite sequence, NT and TSA-NT embryos were substantially demethylated at the blastocyst stage as IVF-derived embryos were demethylated. The in vitro developmental rate (46.6%) of TSA-NT embryos that were individually transferred with TSA-treated cells was higher than that (31.7%) of NT embryos with non-treated cells (P < 0.05). Our findings suggest that the chromatin of a donor cell is unyielding to the reprogramming of DNA methylation during preimplantation development, and that alteration of the epigenetic state of donor cells may improve in vitro developmental competence of cloned embryos.</P>
Inheritable Histone H4 Acetylation of Somatic Chromatins in Cloned Embryos
Wee, Gabbine,Koo, Deog-Bon,Song, Bong-Seok,Kim, Ji-Su,Kang, Man-Jong,Moon, Seung-Ju,Kang, Yong-Kook,Lee, Kyung-Kwang,Han, Yong-Mahn American Society for Biochemistry and Molecular Bi 2006 The Journal of biological chemistry Vol.281 No.9
Behaviors of ATP-dependent chromatin remodeling factors during maturation of bovine oocytes in vitro
Wee, Gabbine,Shin, Sang-Tae,Koo, Deog-Bon,Han, Yong-Mahn JOHN WILEY & SONS LTD 2010 MOLECULAR REPRODUCTION AND DEVELOPMENT Vol.77 No.2
<P>The mammalian oocyte undergoes dynamic changes in chromatin structure to reach complete maturation. However, little known is about behaviors of ATP-dependent chromatin remodeling factors (ACRFs) during meiosis. Here, we found that respective ACRFs may differently behave in the process of oocyte maturation in the bovine. All ACRFs interacted with oocytic chromatin at the germinal vesicle (GV) stage. Mi-2 and hSNF2H disappeared from GV-chromatin within 1 hr of in vitro culture whereas Brg-1 and BAF-170 were retained throughout germinal vesicle break down (GVBD). Brg-1 was localized on the condensed chromatin outside, whereas BAF-170 was entirely excluded from condensed chromatin. Thereafter, Brg-1 and BAF-170 interacted with metaphase I and metaphase II chromosomes. These results imply that Mi-2 and hSNF2H may initiate the meiotic resumption, and Brg-1 and BAF-170 may support chromatin condensation during meiosis. In addition, DNA methylation and methylation of histone H3 at lysine 9 (H3K9) seem to be constantly retained in the oocyte chromatin throughout in vitro maturation. Inhibition of ACRF activity by treatment with the inhibitor apyrase led to retarded chromatin remodeling in bovine oocytes, thereby resulting in poor development of fertilized embryos. Therefore, these results indicate that precise behaviors of ACRFs during meiosis are critical for nuclear maturation and subsequent embryonic development in the bovine. Mol. Reprod. Dev. 77: 126–135, 2010. © 2009 Wiley-Liss, Inc.</P>