PARENT SESSION

(T695) METHYLATION STATUS OF PUTATIVE DMRs OF PORCINE IGF2 AND H19.

Han, Dong Wook¹, Im, Young Bin¹, Gupta, Mukesh Kumar¹, Uhm, Sang Jun¹, Lee, Hoon Taek¹, ¹ Konkuk University, Seoul, Korea

ABSTRACT- DNA methylation controls various developmental processes and its perturbations in various genomic regions especially imprinted genes are assumed to be responsible for abnormalities observed in fetuses and offspring derived by in vitro techniques such as somatic cell nuclear transfer. Insulin-like growth factor 2 (IGF2) and H19 are reciprocally imprinted genes that are evolutionally conserved in various species such as sheep, human and mice and harbor differentially methylated regions (DMRs) which are targets for analyzing the genomic imprinting in nuclear transferred embryos and cancerous cells. However, information on DMRs and imprinting status of these genes in porcine species is not available. Therefore, the aim of the present study was to identify putative DMRs of porcine IGF2-H19 loci and to explore its genomic imprinting by analyzing their DNA methylation status. Genomic DNA were extracted from porcine epididymal spermatozoa and in vitro matured oocytes and treated with sodium bisulfite. Putative DMRs on IGF2-H19 were identified based on comparative sequencing with human and mice genomic data and PCR primers were designed to amplify these putative DMRs by bisulfite sequencing polymerase chain reaction (BS-PCR) which were then subcloned and sequenced. Analysis of results revealed that, as in human and mice, porcine H19 DMRs show monoallelic methylation on sperm. Similarly, DMR2 of porcine IGF2 gene was predominantly methylated in paternal allele also but this was not the case with DMR1 which remained biallelically unmethylated at the CpG islands. To gain more insight, we further investigated upstream region of putative DMR1 of IGF2 as a likely candidate and found its methylation pattern to be consistent with those of other species. Our data thus, suggest the existence of genomic imprinting at porcine IGF2-H19 loci similar to other farm animals and provides a platform for examining the DNA methylation profile of DMRs of these imprinted genes during clonal development as one means of addressing nuclear reprogramming besides analyzing the mechanism of loss of imprinting (LOI) in cancerous cells in porcine species. This work was supported by a grant from BioGreen 21 Program, Rural Development Administration, Republic of Korea.

KEY WORDS: IGF2-H19, differentially methylated regions (DMR), reprogramming, porcine