Giraldo Angelica M, Lynn John W, Purpera Megan N, Godke Robert A, Bondioli Kenneth R
Department of Animal Sciences, Louisiana State University Agricultural Center, Baton Rouge, Louisiana 70803, USA.
Mol Reprod Dev. 2007 Dec;74(12):1514-24. doi: 10.1002/mrd.20740.
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.
有证据表明，核移植（NT）胚胎无法正常发育至少部分可归因于使用分化的细胞核作为供体核质体。据推测，由于体外培养过程中DNA甲基化和组蛋白乙酰化模式的变化，同一细胞系的群体倍增（PDs）之间克隆胚胎的囊胚产生和发育至足月可能会有所不同。本研究的目的是确定染色质重塑蛋白DNA甲基转移酶-1（Dnmt1）、甲基CpG结合蛋白-2（MeCP2）和组蛋白脱乙酰酶-1（HDAC1）的基因表达模式，此外，还测量不同PDs下牛成纤维细胞的DNA甲基化和组蛋白乙酰化水平。从四个50天大的胎儿中建立了牛成纤维细胞系。在PDs 2、7、15、30、45和70时分析Dnmt1、MeCP2、HDAC1、甲基化DNA和乙酰化组蛋白的相对水平。使用Q-PCR检测Dnmt1、HDAC1和MeCP2的RNA水平。通过分别用抗5-甲基胞嘧啶和抗乙酰化组蛋白H3抗体孵育固定细胞来确定甲基化DNA和乙酰化组蛋白的总体水平。细胞用二抗标记，用碘化丙啶复染并通过流式细胞术分析。这些数据表明，参与表观遗传修饰的染色质重塑蛋白mRNA在体外培养过程中发生了改变。体外细胞的甲基化DNA和乙酰化组蛋白模式随培养时间而变化。随后将这些细胞用于核移植将有助于深入了解这些表观遗传修饰如何影响重编程。
