In mammals, epigenetic marks on the X chromosomes are involved in dosage compensation. Specifically, they are required for X chromosome inactivation (XCI), the random transcriptional silencing of one of the two X chromosomes in female cells during late blastocyst development. During natural reproduction, both X chromosomes are active in the female zygote. In somatic-cell cloning, however, the cloned embryos receive one active (Xa) and one inactive (Xi) X chromosome from the donor cells. Patterns of XCIhave been reported normal in cloned mice, but have yet to be investigated in other species. We examined allele-specific expression of the X-linked monoamine oxidase type A (MAOA) gene and the expression of nine additional X-linked genes in nine cloned XX calves. We found aberrant expression patterns in nine of ten X-linked genes and hypomethylation of Xist in organs of deceased clones. Analysis of MAOA expression in bovine placentae from natural reproduction revealed imprinted XCI with preferential inactivation of the paternal X chromosome. In contrast, we found random XCI in placentae of the deceased clones but completely skewed XCI in that of live clones. Thus, incomplete nuclear reprogramming may generate abnormal epigenetic marks on the X chromosomes of cloned cattle, affecting both random and imprinted XCI.

Cloning by nuclear transfer from adult somatic cells is a remarkable demonstration of developmental plasticity. When a nucleus is placed in oocyte cytoplasm, the changes in chromatin structure that govern differentiation can be reversed, and the nucleus can be made to control development to term.

BackgroundReal-time PCR is an efficient tool to measure transcripts and provide valuable quantitative information on gene expression of preimplantation stage embryos. Finding valid reference genes for normalization is essential to interpret the real-time PCR results accurately, and understand the biological dynamics during early development. The use of reference genes also known as housekeeping genes is the most widely applied approach. However, the different genes are not systematically compared, and as a result there is no uniformity between studies in selecting the reference gene. The goals of this study were to compare a wide selection of the most commonly used housekeeping genes in mouse oocytes and preimplantation stage embryos produced under different culture conditions, and select the best stable genes for normalization of gene expression data.ResultsQuantitative real time PCR method was used to evaluate 12 commonly used housekeeping genes (Actb, Gapdh, H2afz, Hprt, Ppia, Ubc, Eef1e1, Tubb4, Hist2h2aa1, Tbp, Bmp7, Polr2a) in multiple individual embryos representing six different developmental stages. The results were analysed, and stable genes were selected using the geNorm software. The expression pattern was almost similar despite differences in the culture system; however, the transcript levels were affected by culture conditions. The genes have showed various stabilities, and have been ranked accordingly.ConclusionCompared to earlier studies with similar objectives, we used a unique approach in analysing larger number of genes, comparing embryo samples derived in vivo or in vitro, analysing the expression in the early and late maternal to zygote transition periods separately, and using multiple individual embryos. Based on detailed quantification, pattern analyses and using the geNorm application, we found Ppia, H2afz and Hprt1 genes to be the most stable across the different stages and culture conditions, while Actb, the classical housekeeping gene, showed the least stability. We recommend the use of the geometric averages of those three genes for normalization in mouse preimplantation-stage gene expression studies.