鄭登貴2006-07-262018-06-292006-07-262018-06-292000http://ntur.lib.ntu.edu.tw//handle/246246/16430本研究計畫旨在探討，源自豬隻發情配種後不同生理階段輸卵管上皮細胞之基因轉譯物，對於豬胚早期發育之調控機制。試驗首先針對體外培養系統中添加轉型生長因子-α(TGF-a)，對豬之胚源性基因表現的影響情形;亦即將處於早期4-細胞階段(early 4-cellstage)之豬胚，置於每ml 培養液中含有20ngTGF-a之培養條件下，經3 小時之體外培養後，再抽取其Total RNA 進行基因差異表現分析。試驗結果證明，此等早期豬胚中有二個基因pTIG-1 及pTIG-2，其表現分別有賴TGF-a之誘發，始克有濟;蓋豬胚之於培養液中未添加TGF-a而被培養者，毫無pTIG-1 及pTIG-2 之表現可言。進一步試驗乃針對pTIG-1 & pTIG-2 之完整cDNA 進行選殖與定列，俾提供往後深入探討該等基因之分生特性，及其對於早期胚胎發育可能扮掩之功能角色;為謀順利達成此一目標，本研究首先乃將源自為數約100 個處於4-細胞階段之豬胚所抽取之 Total RNA， 成功構築於ClontechÒ 之lTriplEx2 載體上，並證明由此所建立之cDNA 基因庫，其力價達1X106;進一步選用pTIG-1 中長度為194 bp 之基因片段為探針，已自前述建立之cDNA 基因庫中，成功篩選獲得pTIG-1 cDNA 之選殖株及次選殖株。針對此等選殖株進行定序分析結果得，證明完整之pTIG-1 cDNA 序列全長為3280 bp，可轉譯出 500 個胺基酸;刻除藉由表現載體製被備該基因之部份蛋白質抗原，俾供後續進行功能性分稀之使用外，並亦嘗試針對該基因進行其起動子序列之選殖與定序，俾深入瞭解其對豬早期胚發育之分子調控機制。In a previous study, we demonstrated the expression of transforming growth factor-a (TGF-a) gene in the epithelium of porcine oviductal at a time corresponding to 4-cell stage (Chang et al., 2000). Here we report on the pTIG-1 (porcine TGF-a induced gene-1) was induced in the porcine four cell embryo within 3 hours of TGF-a treatment (20 ng/ml) using mRNA differential display. By further RT-PCR using these specific primers, we obtain the pTIG-1 mRNA was absent in in vivo developed early four cell embryos but was detected in eight cell and 16-cell up to blastocyst stage embryo in vivo. However, TGF-a treatment of in vitro cultured early four cell embryos resulted in the appearance of pTIG-1 transcripts. In situ hybridization analysis demonstrated that the pTIG-1 was expressed late four cell embryos. Sequencing the full-length pTIG-1 cDNA clone identified one open reading frames of 3280 bp which encode 500 amino acids using cDNA library screen. Sequence computer analysis display that the nucleotide sequence of the pTIG-1 gene was homologous to the mouse Mo25 (mMo25) (Miyamoto et al.,1993). The predicated amino acid sequence revealed that the pTIG-1 might encode a Ca2+ binding protein like as mMo25. In this study, we further investigated the role of TGF-a on early embryogenesis using an in vitro culture system and mRNA differential display in pre-implantation embryos. Indeed, we identified a candidate gene, pTIG-1, the expression of which seemed to be modulated by TGF-aapplication/pdf32189 bytesapplication/pdfzh-TW國立臺灣大學動物科學技術學系暨研究所reporthttp://ntur.lib.ntu.edu.tw/bitstream/246246/16430/1/892313B002049.pdf