Evidence indicates that failure of nuclear transfer (NT) embryos to develop 
normally can be attributed, at least partially, to the use of a differentiated 
cell nucleus as the donor karyoplast. It has been hypothesized that blastocyst 
production and development to term of cloned embryos may differ between 
population doublings (PDs) of the same cell line as a consequence of changes in 
DNA methylation and histone acetylation patterns during in vitro culture. The 
objective of this study was to determine gene expression patterns of the 
chromatin remodeling proteins DNA methyltransferase-1 (Dnmt1), methyl CpG 
binding protein-2 (MeCP2), and histone deacetyltransferse-1 (HDAC1), in 
addition, to measuring levels of DNA methylation and histone acetylation of 
bovine fibroblast cells at different PDs. Bovine fibroblast cell lines were 
established from four 50-day fetuses. Relative levels of Dnmt1, MeCP2, HDAC1, 
methylated DNA, and acetylated histone were analyzed at PDs 2, 7, 15, 30, 45, 
and 70. RNA levels of Dnmt1, HDAC1, and MeCP2 were examined using Q-PCR. Global 
levels of methylated DNA and acetylated histone were determined by incubation of 
fixed cells with an anti-5-methylcytidine and anti-acetyl-histone H3 antibody, 
respectively. Cells were labeled with a second antibody, counter-stained with 
propidium iodide and analyzed by flow cytometry. These data demonstrate that 
chromatin remodeling protein mRNAs involved in epigenetic modifications are 
altered during in vitro culture. Methylated DNA and acetylated histone patterns 
of in vitro cells change with time in culture. Subsequent use of these cells for 
NT will provide insight as to how these epigenetic modifications affect 
reprogramming.