New adenovirus vectors for protein production and gene transfer
New adenovirus vectors for protein production and gene transfer
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DOI：
10.1023/a:1008013211222
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发表时间：
1998-01-01
期刊：
影响因子：
2.2
通讯作者：
Langelier, Y
中科院分区：
文献类型：
--
作者：
Massie, B;Mosser, DD;Langelier, Y
基于两个新的腺病毒表达盒，我们已经构建了一系列的Ad转移载体，用于过表达一个或两个基因，无论是在双顺反子配置或单独的表达盒。在载体中包含绿色或蓝色荧光蛋白加速腺病毒重组体的产生，并通过允许基因转移和表达的容易定量而促进体外和体内基因的功能表征。利用我们优化的四环素调节启动子（TR 5），我们已经产生了表达蛋白质的重组腺病毒，所述蛋白质是细胞毒性的或干扰腺病毒复制，其水平为总细胞蛋白质的10-15%。无细胞毒性的蛋白质可以以大于总细胞蛋白质的20%的水平产生。同样，这些水平的蛋白质生产可以在有或没有腺病毒复制的情况下实现。该产率类似于用我们优化的人巨细胞病毒立即早期启动子-增强子（CMV 5）在非互补细胞系中组成型蛋白表达所能获得的产率。使用绿色荧光蛋白作为报告基因，我们已经表明，pAdCMV 5衍生的腺病毒载体在互补293细胞中表达的蛋白质比含有标准巨细胞病毒启动子的腺病毒载体多约6倍，在非互补HeLa细胞中多约12倍。此外，掺入一系列载体中的绿色荧光蛋白的红移变体比S65 T突变体的荧光高12倍，使得在低得多的表达水平下检测报告蛋白成为可能。
Based on two new adenovirus expression cassettes, we have constructed a series of Ad transfer vectors for the overexpression of one or two genes either in a dicistronic configuration or with separate expression cassettes. Inclusion of the green or blue fluorescent protein in the vectors accelerates the generation of adenovirus recombinants and facilitates the functional characterization of genes both in vitro and in vivo by allowing easy quantification of gene transfer and expression. With our optimized tetracycline-regulated promoter (TR5) we have generated recombinant adenoviruses expressing proteins, that are either cytotoxic or which interfere with adenovirus replication, at levels of 10-15% of total cell protein. Proteins that are not cytotoxic can be produced at levels greater than 20% of total cell protein. As well, these levels of protein production can be achieved with or without adenovirus replication. This yield is similar to what can be obtained with our optimized human cytomegalovirus-immediate early promoter-enhancer (CMV5) for constitutive protein expression in non-complementing cell lines. Using the green fluorescent protein as a reporter, we have shown that a pAdCMV5-derived adenovirus vector expresses about 6-fold more protein in complementing 293 cells and about 12-fold more in non-complementing HeLa cells than an adenovirus vector containing the standard cytomegalovirus promoter. Moreover, a red-shifted variant of green fluorescent protein incorporated in one series of vectors was 12-fold more fluorescent than the S65T mutant, making the detection of the reporter protein possible at much lower levels of expression.