Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21570
標題: 複製豬基因體組中銘印基因上遺傳性修飾異常之研究
The Aberrant Epigenetic Modification of Imprinting Genes in Cloned Swine's Genomes
作者: 沈志傑
Shen, Chih-Jie
關鍵字: imprinting gene;銘印基因;reprogramming process;epigenetic modification;cloned swine;nuclear transfer;differentially methylated region;再程式化作用;上遺傳性修飾;複製豬;核轉置;差異性甲基化片段
出版社: 生命科學系
摘要: 
在哺乳類基因體組中,調控著基因中重要的轉錄訊息而影響基因功能中最主要的因子就是上遺傳性修飾中的DNA甲基化修飾。基因體組上的甲基化修飾模式於已分化的體細胞中是非常穩定且具有可遺傳性的。而在胚胎發育時則有兩個例外的時機點,一個是始基生殖細胞生成的時候,另一個則是合子形成的時候,在這兩個時機點中,其甲基化會經過再程式化的過程,來使得基因體組整體恢復跟親源染色體相同的甲基化模式。銘印基因則是維持胚胎正常生長及發育的重要關鍵,而銘印基因中受甲基化控制之差異性甲基化片段便控制著基因的表現。在利用體細胞核轉置的複製動物中,其胚胎發育有異常、有多發性的器官缺陷、存活率也很低。而存活下來的複製動物個體之銘印基因上的DMR區亦常見有不適當之甲基化修飾,咸認為與銘印基因之異常再程式化有關。在本研究中,利用耳朵纖維母細胞轉置於去核的卵中所產製的四隻複製豬成體為材料,我們利用南方轉漬法、MS-PCR、COBRA、亞硫酸鈉定序等方法來確認在H19、Igf2、Igf2及Ins中的差異性甲基化片段(DMR)是否有甲基化異常。實驗結果顯示在四隻複製豬個體中的四個銘印基因之DMR確有明顯的甲基化異常發生,且好發於各種器官中。本試驗證明了在複製動物成體留存著從胚胎時期即異常之甲基化模式,且推測可能影響其成體後的發育,也可利用本試驗的模式來研究其他複製豬個體,繼續累積足夠的證據來找出核轉置胚存活率低下的關鍵點為何。

DNA methylation is a major epigenetic modification of the genome that regulates crucial aspects of gene function. Genomic methylation patterns in somatic differentiated cells are generally stable and heritable. However, in mammals there are at least two developmental periods, germ cells and preimplantation embryos, in which methylation patterns are reprogrammed genome wide and generating cells with a broad developmental potential. The recent success of somatic cell cloning in mammals gives promise to applications such as species preservation, livestock propagation , and cell therapy for medical treatment. But cloning by nuclear transfer (NT) has been riddled with difficulties: most clones die before birth and survivors frequently display growth abnormalities. The cross-species similarity in abnormalities observed in cloned fetuses/animals leads us to suspect the fidelity of epigenetic reprogramming of the donor genome. In this study, four cloned pigs created by whole cumulus cell nuclear transfer with short life span and multiple organ defects were used as epigenetic experimental materials, and especially defect in the heart and bone. The Southern blot, methylation-specific PCR, combined bisulfite and restriction assay (COBRA), and bisulfite sequencing were applied to search the aberrant methylation in different imprinted gene loci in these cloned pig genomes. Our data revealed that loss of imprinting (LOI) phenomenon was frequently appeared in H19, Igf2, Ins and Igf2r imprinted loci in several tissues of these cloned sows. These observation indicate that defects in epigenetic inheritance arise very early during clonal development, and that epigenetic information associated with imprinted genes is not faithfully retained in the majority of cloned adults.
URI: http://hdl.handle.net/11455/21570
Appears in Collections:生命科學系所

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