Development of stable and regulated gene expression system for gene therapy

在基础遗传学研究中，控制哺乳动物细胞中的转基因表达是基因治疗和基因功能研究的重要内容。永久和更高水平的基因表达也是可取的。引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。在这项研究中，我们通过构建一系列质粒和重组腺病毒载体，研究了常用的调控元件（如启动子/增强子、内含子和多腺苷化信号（P(A)）序列）的有效组合，这些质粒和重组腺病毒载体仅在被评估的特定序列元件上存在差异。在所测试的几种启动子/增强子中，含有人巨细胞病毒立即早1基因（CMV）增强子和含有β-肌动蛋白内含子序列的鸡β-肌动蛋白启动子的杂交CA启动子/增强子，以及含有最大的CMV内含子（int . More ron A）的改良型CMV启动子/增强子在体外和体内均产生最高水平的表达。发现P(A)序列对转基因表达有显著影响。这些比较分析可为基因治疗、疫苗研制和基因转移实验的载体构建提供系统的参考。接下来，我们使用含有腺相关病毒（AAV）成分的质粒检测基因的稳定表达。我们用带有AAVITR标记的含有新霉素耐药基因的质粒转染新霉素耐药细胞，观察其集落形成的效率。转染含有AAVITR和Rep78（aav衍生蛋白表达质粒）的质粒后，新霉素耐药细胞的菌落数量远高于未表达AAVITR和Rep78的质粒，这表明Rep78蛋白有助于外源基因整合到宿主基因组中。我们现在正将这项技术应用于将基因组外源DNA整合到宿主基因组中。该系统利用基因组DNA实现了基因表达的稳定调控。少

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