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標題: 探討體細胞核轉置動物之銘印基因上位遺傳異常修飾作用
Epigenetic Aberration of Imprinting Genes in Somatic Nuclear Transferred Cloning Bovine Genomes
作者: 林巧絜
Lin, Chiao-Chieh
關鍵字: Imprinting Genes;探討體細胞核轉置動物;Somatic Nuclear Transferred Cloning Bovine;銘印基因
出版社: 生命科學系所
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已知哺乳類動物體內,基因體上位修飾再程序化系統存於初級配子細胞與合子細胞內,並於早期胚胎發育過程中,為維繫調控不同胚期之基因功能與表現之關鍵因子。然體細胞核轉置複製技術常伴隨供核細胞之再程序不當調控現象,進而影響其產置效率與複製動物出生前後之存活率。其中部分特定去甲基化因子恐導致組織間基因表現缺失而影響正常生理功能。本篇論文中,其六頭複製牛皆取正常荷蘭母牛之耳朵纖維母細胞為其供核細胞源所產製,惟此等複製牛皆呈極短之生命週期與多重器官嚴重缺失現象。然此等重大缺失是否與銘印基因異常調控有關?本篇論文主要針對胚胎發育過程中,維持胎兒與胎盤正常生長發育之IGF-2與H19兩種銘印基因與主導X染色體失活機制之Xist基因,進行正常牛與複製牛各組織間COBRA分析,並搭配亞硫酸鈉定序檢視各目標片段中,所夾擊之CpG位點DNA甲基化修飾程度。結果顯示,確存有不同複製牛各組織間,不同程度之IGF-2, Xist與H19等基因銘印消逝之異常現象。然若進一步探究複製牛NTG-2之IGF-2, Xist與H19等銘印基因mRNA表現量則發現,IGF-2 與H19廣幅性過表現於肝臟、下大靜脈、耳朵、皮膚與子宮等組織。其中IGF-2於NTG-2之子宮與肝臟組織相較於正常牛者高出約1,200及800倍之多;而H19於NTG-2之肝臟組織甚至較正常牛者高出34,000倍左右。藉由相關試驗證實,相信此等鉅幅過表現之IGF-2與H19,恐與NTG-2多重器官發育缺失有關。縱歸上述,體細胞核轉置複製動物之銘印基因異常DNA甲基化修飾與再程序化調控失序,應是導致其發育不全抑或出生前後高死亡率之主因。

In mammals, genome-wide epigenetic reprogramming systems exist in primordial germ cells and zygote. It plays a crucial role in regulating genome functions at critical stages of embryo development and confers stability of gene expression during mammalian development. Inappropriate reprogramming of donor nuclei in somatic cell resulted in neonatal death and preimplantation defects during animal cloning technique. Specific demethylation events in differentiated tissues could then lead to further changes in gene expression as needed. In this study, six cloned bovines created by ear fibroblast nuclear transfer with short life span and multiple organ defects were used as the experimental materials. We focus on three imprinting genes included two growth factor genes IGF-2, H19 and Xist (X chromosome inactive regulated gene) by combined bisulfite and restriction assay (COBRA). Bisulfite sequencing were also applied to analyze the aberrant CpG sites in different imprinted genes loci in these cloned bovine genomes. Our data show that loss of imprinting (LOI) phenomenon was frequently appeared in IGF-2, Xist and H19 imprinted loci in several tissues of these cloned bovines. Furthermore, we detected the cloned bovine NTG-2 expression of these imprinting genes with quantitative real time PCR (Q-PCR). The data suggested that IGF-2 and H19 are extremely overexpressed in liver, vein, ear, skin and uterus. The IGF-2 mRNA expression in uterus and liver of NTG-2 cow are almost 1,200 and 800 folds compared to WT cow. The H19 expression level in liver of NTG-2 also exhibited a 34,000-fold higher than that of WT cow. According to our research, the extensively overexpression of IGF-2 and H19, probably were the major reason to cause the cloned bovine NTG-2 not only organ defects, but also aberrant embryonic development. In conclusion, the death of clones may be due to aberrant DNA methylation at the loci of imprinting genes and disruption of incompletely reprogrammed after nuclear transfer.
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