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Development of stable and regulated gene expression system for gene therapy

开发用于基因治疗的稳定且受调控的基因表达系统

基本信息

批准号：
12672145
负责人：
HAYAKAWA Takao
金额：
$ 2.18万
依托单位：
National Institute of Health Sciences
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12672145/
关键词：
gene therapy adenovirus vector adeno-associated virus vector gene requlation plasmid vector

项目摘要

In basic genetic research, control oftransgene expression in mammalian cells should be desirable forgene therapyand the studyofgene function. Permanentand higher levels of gene expression should be also preferable. The expression levels of the introduced gene depend mostly on the strength oftranscriptional regulatory elements and the transduction efficiency of the gene transfer vector.In this study, we investigated the effective combination of commonly-used regulatory elements, such as the promoter/enhancer, intron, and polyadenylation signal (P(A)) sequence by constructing a series of plasmids and recombinant adenovirus vectors that differed only in the particular sequence element being evaluated. Of the several promoter/enhancers that were tested, hybrid CA promoter/enhancer containing human cytomegalovirus immediate-early 1 gene (CMV) enhancer and chicken β-actin promoterwith the β-actin intron sequence, and the improved CMV promoter/enhancer containing the largest intron of CMV(int … More ron A) produced the highest levels of expression both in vitro and in vivo. P(A)sequences were found to have significant effects on transgene expression. These comparative analyses could provide a systematic reference for the development of vector construction for gene therapy, vaccine development, and gene transfer experiments.Next, we examined the stable gene expression using the plasmids containing the components derived from adeno-associated virus (AAV). We examined the efficiency of colony formation of the neomycin resistant cells which were transfected with the plasmid containing neomycin resistant gene franked with AAVITR (inverted terminal repeat). Number of colony of neomycin resistant cells transfected with the plasmid with AAVITR and Rep78(AAVderived proteinj-expressing plasmid was much higher than that with the plasmid without AAVITRand Rep78expressing plasmid, suggesting that Rep78 protein assisted the integration of the foreing gene intothe host genome. We are now applying this techiniqes intothe integration of the genomic foreign DNA into the host genome. This system using genomic DNA could get stable and regulated gene expression. Less

在基础遗传学研究中，控制转基因在哺乳动物细胞中的表达是基因治疗和基因功能研究的需要。永久和更高水平的基因表达也应该是更可取的。导入基因的表达水平主要取决于转录调控元件的强度和基因转移载体的转导效率。在本研究中，我们通过构建一系列仅在特定序列元件上存在差异的质粒和重组腺病毒载体，研究了启动子/增强子、内含子和多聚腺苷信号(P(A))序列等常用调控元件的有效组合。在测试的几个启动子/增强子中，含有人巨细胞病毒即刻早期1基因(CMV)增强子和鸡β-肌动蛋白启动子的杂交CA启动子/增强子带有β-肌动蛋白内含子序列，以及含有CMV最大内含子(INT…)的改进的CMV启动子/增强子更多的RON A)在体外和体内均产生最高水平的表达。P(A)序列对转基因表达有显著影响。这些比较分析可以为基因治疗、疫苗开发和基因转移实验的载体构建提供系统的参考。接下来，我们使用含腺相关病毒(AAV)组件的质粒检测了稳定的基因表达。我们检测了以AAVITR(反向末端重复序列)标记的新霉素抗性基因表达载体对新霉素耐药细胞集落形成的影响。含AAVITR和Rep78(AAV衍生蛋白)表达载体的新霉素耐药细胞克隆数明显高于不含AAVITR和Rep78表达载体的细胞克隆数，提示Rep78蛋白有助于外源基因整合到宿主基因组中。我们现在正在将这项技术应用于将基因组中的外源DNA整合到宿主基因组中。该系统利用基因组DNA可以获得稳定的、可调控的基因表达。较少