鄭登貴2006-07-262018-06-292006-07-262018-06-292002http://ntur.lib.ntu.edu.tw//handle/246246/16447本研究旨在以小鼠為模式，探討母源性與胚源性基因對於早期胚發育之分子調控機制。試驗首先應用mRNA 分示法(mRNAdifferential display)，針對小鼠之成熟卵母細胞與處於早期發育階段(2- ~ 8-細胞) 之小鼠胚，比較其對於母源性基因及胚源性基因之表現的差異性，再就彼等表現具有差異性之特定基因分別進行選殖及定序，並針對其上游序列進行測試分析，俾瞭解其表現之分子調控機制。試驗結果合計獲得 20 個表現具有生理階段特異性之基因片段，包括3 個係源自母源性基因所表現者，蓋其轉錄物僅在成熟母細胞及2-細胞階段之早期胚中可被測得;及17 個係源自胚源性基因所表現者，蓋其轉錄物並未出現於成熟母細胞中，而需俟胚發育達2- ~ 4-細胞甚或更後期之階段，始克被測得。在前述3 個母源性基因之一，名為mOSG (mouse oocyte specific gene)者，經完成其全長cDNA 之選殖與定序結果，並證明係一長度為 875 bp，可轉譯出一個含有105 胺基酸之蛋白質。在後續試驗中，更經由反轉錄聚合連鎖反應 (RT-PCR)及免疫定位 (immuno-localization) 技術之配合應用，證實mOSG 之基因轉錄物確係僅呈現於成熟母細胞及2-細胞階段之早期胚中者，惟其轉譯出之蛋白質則遲至 8-細胞階段始被降解完全。此外，配合DNA 序列網羅策略(DNAsequencing shotgun strategy)與表現性序列標株(expressional sequencing tag clone,EST-clone)之聯合應用，針對處於埋殖前發育階段之小鼠胚，嘗試篩選彼等表現具有發育階段特異性之胚源性鋅指蛋白 (zinc fingerproteins)轉錄因子;結果並成功獲得一個名為mZFG-1 (mouse zinc finger gene-1)之鋅指蛋白基因。進一步針對該基因完成其全長cDNA之選殖、定序及其表現模式(expression profile)之鑑定。試驗結果證明該基因轉錄物全長為1,338 bp;鑑於mZFG-1S 之基因轉錄物僅當成熟卵母細胞完成受精作用後始克被表現，且表現量係隨著卵裂之進展而增加，而於到囊胚階段時其表現量復又轉趨減少，顯示該基因不僅確係屬於胚源性基因，且其表現頗與胚之發育階段有密切關係。In this present study the expression of mouse maternal and zygotic genes was compared and identified using two strategies based on differential display and expression sequences tags (EST) against samples from freshly ovulated mouse oocytes and 8-cell embryos, respectively. For mRNA differential display studies, a total 30 combinations of arbitrary with anchor primers were used and results appeared that of the 20 genes identified, 17 zygotic genes were found to be specifically expressed in embryos only if they had reached to 2- ~ 4-cells or even 2 at much advanced stages. Whereas, expression of the other three genes were characterized as the maternal ones found only in those ovulated oocytes before ferilization. One of the mOSG (mouse oocyte specific gene; Gene bank accession number: AF313913) cDNA was detected only in mouse oocytes and showed a high sequencing homology with the mouse OM2a and OM2b, and may be a novel member of this gene family. By further rapid amplification 5’-end (RACE) techniques, we obtained the full-length cDNA with 875 bp to encode 105 amino acids. RT-PCR and Northern blotting analyses demonstrated that the mOSG mRNA was expressed merely in the mouse ovary. Further localization of transcripts using in situ hybridization confirmed that expression of mOSG occurred only in matured oocytes in antrum follicles. On the other hand, using the mouse EST-based primer-sets for RT-PCR, the mZFG-1 (Zinc finger protein-1) gene was identified and this gene was further confirmed being expressed specifically in mouse pre-implantation embryos (between 1-cell and blastocyst stages). Based on the fact that increase in amount of mZFG-1 gene transcripts along the stages of embryos from 1-cell to 8-cells and subsequently followed by decrease in its transcription activity suggested mZFG-1 gene may play an important role related to early development and/or early differentiation events.application/pdf603081 bytesapplication/pdfzh-TW國立臺灣大學動物科學技術學系暨研究所mouse embryomaternal genezygotic genegene expressionreporthttp://ntur.lib.ntu.edu.tw/bitstream/246246/16447/1/902313B002303.pdf