| Summary: | 碩士 === 東海大學 === 畜產與生物科技學系 === 103 === The oocyte-specific linker histone H1 (H1Foo) participates in regulation of gene expression in various steps of embryogenesis. The sequence of full-length H1Foo cDNA has been identified in mice, human, bovine and other non-mammalian species, but not in pig. The objective of this study were to investigate the involvement of porcine H1Foo (pH1Foo) in early embryogenesis by obtaining the full length pH1Foo cDNA and overexpression of pH1F00 in somatic cells. By using 5’-rapid amplification of cDNA end (RACE) method, total RNAs from germinal vesicle (GV)-stage oocytes were used to obtain the full length cDNA. For the one-step reverse transcription-polymerase chain reaction (RT-PCR), RT was performed using pH1Foo gene-specific reverse primer and subsequent PCR was carried out by adding a forward primer containing the adaptor sequence. The resulting PCR fragments were cloned into a TA cloning vector and then sequenced. The full length of pH1Foo cDNA was 1107 bp and 346 amino acids encoded open reading frame. The pH1Foo cDNA was inserted into the eukaryotic expression vector and trensfected into the porcine aortic endothelial cells (PECs). The result of localization of H1Foo-enhanced green fluorescent protein (EGFP) indicated H1Foo protein was located in nucleus. The result of Real-time PCR indicated that the expression of stem cell related genes (Oct4, Nanog and C-myc) were significantly increased in pH1Foo tranfected PECs. In addition, In pH1Foo tranfected PECs, proportion of the G0/G1 phase cells were reduced and the sub-G1 phase cells were increased. In conclusion, the H1Foo protein affected the stem cell related genes (Oct4, Nanog and C-myc) and induced the cell apoptosis.
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