Molecular Structure Of Bovine Gtl2 Gene And DNA Methylation Status Of Dlk1–Gtl2 Imprinted Domain In Cloned Bovines

Somatic cell nuclear transfer(SCNT)has been successfully applied to many mammalian species. Somatic cell nuclear transfer(SCNT)technology has been employed to produce animals of a wide variety of mammalian species. However, many of those that can develop to birth often display a variety of the organ abnormalities. It appears to be a great barrier to efficient cloning. The incomplete reprogramming of donor cell nuclei leading to aberrant expression or lack of expression of some developmentally important genes has been implicated as a primary reason for the low efficiency of SCNT. DNA methylation is a major epigenetic modification of the genome that regulates some crucial aspects of its function.Genomic imprinting is an epigenetic phenomenon that results in an allele-specific expression, depending on its parental origin. Dlk1-Gtl2 imprinted domain are involved in embryonic and placental growth and is conserved among species. Dlk1-Gtl2 imprinted domain has been widely studied in mouse and human. However, little is known in bovine, possibly because of limited appropriate sequences of bovine.In our study, we first isolated the cDNA sequence and found multiple transcript variants occured in bovine Gtl2 gene. In addition, none of the ATGs is consistent with Kozak consensus sequence. So the bovine Gtl2 gene may act as an noncoding RNA. Comparison the Gtl2 cDNA sequences we have obtained and the Dlk1 cDNA sequence in NCBI with bovine genomic sequence, we detected that the Dlk1-Gtl2 imprinted domain spaned probably 110 kb on bovine chromosome 21 and identified the two putative DMRs in Dlk1-Gtl2 imprinted domain.Bisulfite sequencing enables us to obtain the methylation status of every CpG site in the objective fragment with highly authenticity and accuracy. In order to make sure whether the DNA methylation reprogramming is efficient in SCNT animals, we analyzed the DNA methylation status of the two DMRs and Dlk1 5′promoter in lungs of deceased SCNT bovines that died within 48 h after birth and the normal controls. In cloned bovines, Gtl2 DMR exhibited hypermethylation which was similar to controls. However, the methylation status of IG-DMR and Dlk1 5′promoter in clones was significantly different from controls, with severe loss of methylation in IG-DMR and hypermethylation in the Dlk1 5′promoter region. Our data suggested that abnormal methylation patterns of IG-DMR and Dlk1 5′promoter may lead to the abnormal expression of Gtl2 and the aberrant development of lungs of cloned bovines, which consequently may contribute to the low efficiency of SCNT.