Abstract
DNA methylation is a major epigenetic modification in the mammalian genome that regulates crucial aspects of gene function. Mammalian cloning by somatic cell nuclear transfer (SCNT) often results in gestational or neonatal failure with only a small proportion of manipulated embryos producing live births. Many of the embryos that survive to term later succumb to a variety of abnormalities that are likely due to inappropriate epigenetic reprogramming. Aberrant methylation patterns of imprinted genes in cloned cattle and mice have been elucidated, but few reports have analyzed the cloned pig genome. Four surviving cloned sows that were created by ear fibroblast nuclear transfer, each with a different life span and multiple organ defects, such as heart defects and bone growth delay, were used as epigenetic study materials. First, we identified four putative differential methylation regions (DMR) of imprinted genes in the wild-type pig genome, including two maternally imprinted loci (INS and IGF2) and two paternally imprinted loci (H19 and IGF2R). Aberrant DNA methylation, either hypermethylation or hypomethylation, commonly appeared in H19 (45% of imprinted loci hypermethylated vs. 30% hypomethylated), IGF2 (40% vs. 0%), INS (50% vs. 5%), and IGF2R (15% vs. 45%) in multiple tissues from these four cloned sows compared with wild-type pigs. Our data suggest that aberrant epigenetic modifications occur frequently in the genome of cloned swine. Even with successful production of cloned swine that avoid prenatal or postnatal death, the perturbation of methylation in imprinted genes still exists, which may be one of reason for their adult pathologies and short life. Understanding the aberrant pattern of gene imprinting would permit improvements in future cloning techniques.
Highlights
Somatic cell nuclear transfer (SCNT) is the transmission of a differentiated somatic cell nucleus to an enucleated oocyte
We compared the well-known differential methylation regions (DMR) of insulin-like growth factor 2 (IGF2), H19, INS, and IGF2R in the human, bovine, and mouse genomes to identify the counterparts of these DMRs in pig genome
The putative DMR1 of the pig IGF2 gene was predicted to be located in intron 3, and the putative DMR of IGF2 (DMR2) of pig IGF2 was in exon 9 (Figure 1A)
Summary
Somatic cell nuclear transfer (SCNT) is the transmission of a differentiated somatic cell nucleus to an enucleated oocyte. SCNT-cloned mammals usually have a low survival rate due to abortion, neonatal death and postnatal defects. Animals that have been successfully cloned during the past decade include sheep, cattle, goats, pigs, rabbits, mice, cats, and dogs [3,4,5]. A variety of nuclear cell types and stages of oocytes have been tried, the success rate still remains low [6]. Most of surviving clones have physiological problems; for example, large offspring syndrome (LOS) and placental abnormalities have been found in cloned cattle, sheep, and mice [7,8,9]. Understanding aberrant methylation patterns and correcting perturbed epigenetic modification will help improve the health of cloned animals
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