Methylation pattern of H19 Gene at Various Preimplantation Development Stages on In Vitro-Fertilized and Cloned Porcine Embryos

서지정보

· 발행기관 : 한국동물번식학회
· 수록지 정보 : Reproductive & developmental biology / 31권 / 2호
· 저자명 : Young-Bin Im, Dong-Wook Han, Mukesh Kumar Gupta, Sang Jun Uhm, Young Tae Heo, Jin-Hoi Kim, Chankyu Park, Hoon Taek Lee

목차

ABSTRACT INTRIDUSTION MATERIALS AND METHODS oocyte Retrieval and In Vitro Maturation In Vitro Fertilization(IVF) of Oocytes Preparation of Donor Cells for Nuclear Transfer Somatic Cell Nuclear Transfer(SCNT) of Oocytes Comparative Sequencing Genomic DNA Isolation Bisulfite(BS) Treatment and Polymerase Chain Reaction(PCR) Cloning and sequencing Experimental Designs Statistical Analysis RESULTS Sequence Homology Bisulfite Sequencing Analysis of Putative DMRs Bisulfite Sequencing Analysis of Pocine IVF and SCNT Embryos DISCUSSION REFERENCES

영어초록

Insulin-like growth factor II (IGF2) and H19 genes are mutually imprinted genes which may be responsible for abnormalities in the cloned fetuses and offspring. This study was performed to identify putative differentially methylated regions (DMRs) of porcine H19 locus and to explore its genomic imprinting in in vitro fertilized (IVF) and somatic cell nuclear transferred (SCNT) embryos. Based on mice genomic data, we identified DMRs on H19 and found porcine H19 DMRs that included three CTCF binding sites. Methylation patterns in IVF and SCNT embryos at the 2-, 4-, 8～16-cells and blastocyst stages were analyzed by BS (Bisulfite Sequencing)-PCR. The CpGs in CTCF1 was significantly unmethylated in the 2-cell stage IVF embryos. However, the 4- (29.1%) and 8～16-cell (68.2%) and blastocyst (48.2%) stages showed higher methylation levels (p<0.01). On the other hand, SCNT embryos were unmethylayted (0～2%) at all stages of development. The CpGs in CTCF2 showed almost unmethylation levels at the 2-, 4- and 8～16-cell and blastocyst stages of development in both IVF (0～14.1%) and SCNT (0～6.4%) embryos. At all stages of development, CTCF3 was unmethylated in IVF (0～17.3%) and SCNT (0～1.2%) embryos except at the blastocyst stage (54.5%) of IVF embryos. In conclusion, porcine SCNT embryos showed an aberrant methylation pattern comprised to IVF embryos. Therefore, we suggest that the aberrant methylation pattern of H19 loci may be a reason for increased abnormal fetus after embryo transfer of porcine SCNT embryos.

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