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標題: 利用piggyBac轉座子系統由人類胚胎幹細胞產製螢光標記之神經細胞
Generation of fluorescence-tagged neural cells from human embryonic stem cells by using piggyBac transposon system
作者: 沈博文
Shen, Po-Wen
關鍵字: 人類胚胎幹細胞;human embryonic stem cells;piggyBac transposon system
出版社: 生命科學系所
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胚胎幹細胞擁有在體外培養時自我增生,以及可以分化為全身各種細胞型態的能力,因此被認為是可使用於再生醫學及發育研究的最佳素材。此外,人類胚胎幹細胞的基因修改是很重要的工具,其可用於基因功能探討、特定細胞的純化以及移植後的細胞追蹤等。我們的實驗目的是希望建立可產生綠色螢光的人類胚胎幹細胞株,並在其分化成特定種類神經細胞時產生紅色螢光,以用於分化為特定神經細胞的純化。我們使用piggyBac轉座子系統做為鑲嵌外來基因於TW1的轉殖基因方式。在piggyBac的質體上,我們一開始使用了多種的萬能性幹細胞的標記啟動子來驅動G418抗性以及綠色螢光的報導基因,如人類Nanog和不同長度片段的人類Oct4 啟動子。我們發現近端和完整的人類Oct4啟動子可以於未分化的TW1 hESCs,驅動下游neomycin resistant gene和螢光表現。而為了獲得穩定的人類胚胎幹細胞轉殖基因效率,我們使用CAG promoter大量表現piggyBac transposase以提高鑲嵌效能。將帶有transposase和外來基因的piggyBac質體以電穿孔方式送入TW1細胞後, CAG-GFP約有40~50%的螢光表現,而Oct4-GFP的螢光表現約有18%。接著我們使用G418進行四週的抗生素篩選,觀察仍約有近25%的螢光持續表現。為獲得在神經發育的不同階段會有不同螢光表現的人類胚胎幹細胞株,我們先測試以Nestin啟動子來驅動紅色螢光報導基因的質體,並將此質體以電穿孔方式轉殖入已分化12天的TW1細胞中,或以轉染的方式轉殖入human neuroblastoma NT-2細胞中,發現以上兩者皆有紅色螢光的表現。以此為基礎,我進一步在質體Oct4-NeoGFP/pXLBac加入了以Nestin promoter或是αTubulin promoter驅動紅色螢光以標定神經幹細胞。而在將經過測試後,質體Oct4-NeoGFP/Nestin-DsRed/pXLBac可成功在TW1細胞中有綠色的螢光表現

The derivatives of pluripotent embryonic stem cells (ESCs) are considered to be functional and unlimited cell sources for basic developmental studies and clinical regenerative medicine. Genetic targeted human embryonic stem cells will further benefit the exploring of gene function, cell purification, and the cell tracking after transplantation into the tissues. Our purpose is to generate genetic modified hESCs for marking undifferentiated ESCs and specific lineage derivatives. The newly-developed Piggybac transposon system is applied to enhance the DNA integration efficiency in hESCs. We initially observed that 40-50 % TW1 hESCs were transfected using pCAG-GFP plasmid with Amaxa electrotransfection reagents. Several promoter regions, including the human Nanog and different segments of human Oct4 promoter, were cloned into a locus upstream the fused GFP and neomycin resistant genes. Our data revealed that only the proximal and full human Oct4 promoter constitutively drove the GFP expression and rendered the transfected cells survived for 4 weeks after G418 selection. The reporter construction for neural stem cells and mature neural cells, Oct4-NeoGFP/Nestin-DsRed/pXLBac and Oct4-NeoGFP/αTubulin-DsRed /pXLBac, respectively, were electroporated into hESCs and human neuroblastoma NT2 cells for the validation of fluorescent protein expression at specific neural stages. The harvest of the labeled specific neural cells will be applied for biomedical researches, such as developmental studies, functional cell analysis and cell transplantation.
其他識別: U0005-1807201316203100
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