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.

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   REFERENCES

• Hyun Seung Lee(Department of Animal Biotechnology, Animal Resources Research Center/Bio-Organ Research Center, Konkuk University, Seoul 143-701, Korea)
• Sung Ho Lee(Department of Animal Biotechnology, Animal Resources Research Center/Bio-Organ Research Center, Konkuk University, Seoul 143-701, Korea)
• Mukesh Kumar Gupta(Department of Animal Biotechnology, Animal Resources Research Center/Bio-Organ Research Center, Konkuk University, Seoul 143-701, Korea)
• Sang Jun Uhm(Department of Animal Biotechnology, Animal Resources Research Center/Bio-Organ Research Center, Konkuk University, Seoul 143-701, Korea)
• Hoon Taek Lee(Department of Animal Biotechnology, Animal Resources Research Center/Bio-Organ Research Center, Konkuk University, Seoul 143-701, Korea) Corresponding author