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Epigenetic Characteristics of Bovine Donor Cells for Nuclear Transfer: Levels of Histone Acetylation¹

Department of Animal Science/Connecticut Center for Regenerative Biology, University of Connecticut, Storrs, Connecticut 06269

Donor cell type, cell-cycle stage, and passage number of cultured cells all affect the developmental potential of cloned embryos. Because acetylation of the histones on nuclear chromatin is an important aspect of gene activation, the present study investigated the differences in histone acetylation of bovine fibroblast and cumulus cells at various passages and cell-cycle stages. The acetylation was qualitatively analyzed by epifluorescent confocal microscopy and quantitatively by immunofluorescent flow cytometry. Specifically, we studied levels of histone H4 acetylated at lysine 8 and histone H3 acetylated at lysine 18; acetylation at these lysine residues is among the most common for these histone molecules. We also studied levels of linker histone H1 in donor cells. Our results show that stage of cell cycle, cell type, and number of cell passages all had an effect on histone content. Histone H1 and acetyl histone H3 increased with cell passage (passages 5–15) in G₀/G₁- and G₂/M-stage cumulus and fibroblast cells. We also found that acetyl histone H4 was lower in early versus late cell passages (passage 5 vs. 15) for G₀/G₁-stage cumulus cells. In both cell types examined, acetyl histones increased with cell-cycle progression from G₀/G₁ into the S and G₂/M phases. These results indicate that histone acetylation status is remodeled by in vitro cell culture, and this may have implications for nuclear transfer.

¹ This manuscript is a scientific contribution of the Storrs Agricultural Experimental Station at the University of Connecticut and was supported by grants to X.C.T and X.Y.

² Correspondence: X. C. Tian, Agricultural Biotechnology Laboratory, 1392 Storrs Road, U-4243, University of Connecticut, Storrs, CT 06269-4243. FAX: 860 486 8809; xtian{at}canr.uconn.edu

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