Methylation Status of H19 Gene in Embryos Produced by Nuclear Transfer of Spermatogonial Stem Cells in Pig
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서지정보
ㆍ발행기관 : 한국동물번식학회
ㆍ수록지정보 : Reproductive & developmental biology / 35권 / 1호
ㆍ저자명 : Hyun Seung Lee, Sung Ho Lee, Mukesh Kumar Gupta, Sang Jun Uhm, Hoon Taek Lee
ㆍ저자명 : Hyun Seung Lee, Sung Ho Lee, Mukesh Kumar Gupta, Sang Jun Uhm, Hoon Taek Lee
목차
ABSTRACT INTRODUCTION MATERIALS AND METHODS Isolation and Culture of Porcine SSCs Immunohistochemistry Immunocytochemistry Oocyte Retrieval and In Vitro Maturation Somatic Cell Nuclear Transfer Bisulfate Treatment PCR Amplification and Sequencing of Bisulfite-treatedGenomic DNA Reverse Transcriptase-polymerase Chain Reaction Statistical Analyses RESULTS The Histological Analysis of Porcine Testis The Isolation and Characterization of pSSCs Analysis of the Methylation Status in pSSCs and pSSCNTEmbryo DISCUSSION REFERENCES영어 초록
The faulty regulation of imprinting gene lead to the abnormal development of reconstructed embryo after nuclear transfer. However, the correlation between the imprinting status of donor cell and preimplantation stage of embryo development is not yet clear. In this study, to determine this correlation, we used the porcine spermatogonial stem cell (pSSC) and fetal fibroblast (pFF) as donor cells. As the results, the isolated cells with laminin matrix selection strongly expressed the GFRα-1 and PLZF genes of SSCs specific markers. The pSSCs were maintained to 12 passages and positive for the pluripotent marker including OCT4, SSEA1 and NANOG. The methylation analysis of H19 DMR of pSSCs revealed that the zinc finger protein binding sites CTCF3 of H19 DMRs displayed an androgenic imprinting pattern (92.7%). Also, to investigate the reprogramming potential of pSSCs as donor cell, we compared the development rate and methylation status of H19 gene between the reconstructed embryos from pFF and pSSC. This result showed no significant differences of the development rate between the pFFs (11.2±0.8%) and SSCs (13.3±1.1%). However, interestingly, while the CTCF3 methylation status of pFF-NT blastocyst was decreased (36.3%), and the CTCF3 methylation status of pSSC-NT blastocyst was maintained. Therefore, this result suggested that the genomic imprinting status of pSSCs is more effective than that of normal somatic cells for the normal development because the maintenance of imprinting pattern is very important in early embryo stage.참고 자료
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