Genome-Wide Epigenetic Alterations in Cloned Bovine Fetuses1

Abstract

To gain a better understanding of global methylation differences associated with development of nuclear transfer (NT)-generated cattle, we analyzed the genome-wide methylation status of spontaneously aborted cloned fetuses, cloned fetuses, and adult clones that were derived from transgenic and nontransgenic cumulus, genital ridge, and body cell lines. Cloned fetuses were recovered from ongoing normal pregnancies and were morphologically normal. Fetuses generated by artificial insemination (AI) were used as controls. In vitro fertilization (IVF) fetuses were compared with AI controls to assess effects of in vitro culture on the 5-methylcytosine content of fetal genomes. All of the fetuses were female. Skin biopsies were obtained from cloned and AI-generated adult cows. All of the adult clones were phenotypically normal and lactating and had no history of health or reproductive disorders. Genome-wide cytosine methylation levels were monitored by reverse-phase HPLC, and results indicated reduced levels of methylated cytosine in NT-generated fetuses. In contrast, no differences were observed between adult, lactating clones and similarly aged lactating cows produced by AI. These data imply that survivability of cloned cattle may be closely related to the global DNA methylation status. This is the first report to indicate that global methylation losses may contribute to the developmental failure of cloned bovine fetuses.

Review Paper: A Review of the Pathology of Abnormal Placentae of Somatic Cell Nuclear Transfer Clone Pregnancies in Cattle, Sheep, and Mice

“…7 In somatic cell cloning, the donor cell genome is compelled to bypass gametogenesis and thus has a different chromatin structure from that of sperm. Aberrant DNA methylation patterns in SCNT embryos and fetuses 11 suggest that most somatic nuclei fail to be reprogrammed, resulting in aberrant gene expression and abnormal phenotypes, particularly in extraembryonic tissues.…”

Mammalian nuclear transfer

“…To establish a potential correlation between global DNA methylation levels and the developmental potential of cloned embryos, Cezar et al compared the genome-wide methylation status among spontaneously aborted cloned fetuses, live cloned fetuses, and adult clones in bovine (Cezar et al, 2003). When genome-wide cytosine methylation levels were measured by reverse-phase high performance liquid chromatography, they found that a significant number of aborted fetuses lacked detectable levels of 5-methylcytosine.…”

Expression of X‐linked genes in deceased neonates and surviving cloned female piglets

“…However, little is known about this reprogramming process, and the overall success rate of animal cloning using somatic cells remains very low across different animal species, likely due to insufficient epigenetic reprogramming. Faulty or incomplete epigenetic reprogramming has been observed in different stages of development (Cezar et al 2003;Kang et al 2001b;Mann et al 2003). Even in cloned animals that survive to term, different aspects of epigenetic abnormalities are evident, although the majority of these animals appear to be healthy and normal (Archer et al 2003;Li et al 2004;Xue et al 2002).…”