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研究生:賴秀春
研究生(外文):Hishu-Ciun La
論文名稱:以寡核酸進行基因更正-提高更正率之研究
論文名稱(外文):Gene Correction Using Oligonucleotide as Template: Studies of Increasing the Correction Efficiency
指導教授:歐樂君
指導教授(外文):Lo-Chun Au
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:55
中文關鍵詞:基因更正更正率經修飾之單股寡核酸
外文關鍵詞:gene correctioncorrection efficiencymodified single-stranded oligonucleotidedoxorubicinthymidine
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以經修飾之單股寡核酸進行基因更正能將基因體的序列做定點改變,這個技術已於大腸桿菌甚至是動植物的實驗模式裡都證實可行。這個技術在基因治療上有很大的潛力,因為它能直接將致病基因的DNA突變更正,使得更正後的基因能在正常的調控下表現。然而,至今這個技術的基因更正率很低,因而局限了它在基因治療臨床上的應用。所以,為了探討影響基因更正率的因素,我們利用中國倉鼠卵巢細胞建立帶有點突變綠螢光蛋白基因的細胞株。若細胞以經修飾之單股寡核酸做模版,利用錯配對機制將點突變更正後,細胞會發出綠色螢光,也就能利用流式細胞儀來分析螢光細胞的比率,藉以偵測基因更正率。我們測試了幾個可能影響基因更正率的因素,希望能將更正率提高。實驗結果發現,以下二種方式能明顯提升基因更正率二倍:一是在經修飾之單股寡核酸轉染進細胞4小時後,以0.2 uM doxorubicin處理細胞15小時;另一則是以thymidine讓絕大部分的細胞停滯在合成期後,再進行單股寡核酸轉染。此外,我們發現經修飾之單股寡核酸的GC比例愈高,基因更正效果愈好,而且antisense單股寡核酸的基因更正效果都比sense單股寡核酸為佳,這顯示出基因更正偏好發生在DNA的非轉錄股。我們也觀察到帶有二個錯配對之單股寡核酸能同時改變目標基因的二個核苷酸。其次,寡核酸上CpG的甲基化及細胞染色體DNA去甲基化處理均不會提高更正率,這顯示細胞可能不是以DNA甲基化的程度來決定何者為修補模版。以上這些實驗結果將有助我們對基因更正進行的機制與影響因素有更多的了解。
The modified single-stranded oligonucleotide vector (MSSOV) was found to be effective in the targeted alternation of genomic sequence in Escherichia coli, yeast and even in plats and mammals. This strategy has potential for gene therapy, because the corrected gene is heritable and is regulated under its own promoter. However, up to now, the efficiency of gene correction is too low to be applied clinically. To investigate what parameters influence the efficiency of gene correction, we constructed two vectors carrying the mutant GFP gene with different single-base nonsense mutations and transfected them into CHO-K1 cells. Once the nonsense mutation of the GFP gene is corrected, the fluorescence of native GFP can be monitored and the efficiency of correction is calculated using flow cytometry. Several parameters, which are possible to affect the correction efficiency, were examined, resulting that the following two procedures could significantly improve correction efficiency more than two folds: (1) cells were transfected with MSSOV for 4h, and subsequently treated with 0.2 uM doxorubicin for another 15h; (2) cells were synchronized at S phase by thymidine block, and then transfected with MSSOV. Furthermore, MSSOV with higher GC content resulted in correction efficiency. Strand bias was observed that correction efficiency of antisense MSSOV was higher than that of Sense MSSOV, indicating that gene correction preferred non-transcribed strand. Moreover, MSSOV carrying two mismatches would frequently trigger alternations of both nucleotides in the target gene. Methylation of CpG on MSSOV and hypomethylation of chromosome DNA carried by 5-aza-cytidine did not increase the correction rate, implicating that the methylation status is not involved in choice of template. The aforementioned results may also be helpful to elucidate mechanism of gene correction.
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