Vectors for inducible expression of toxic gene products in bloodstream and procyclic Trypanosoma brucei
Vectors for inducible expression of toxic gene products in bloodstream and procyclic Trypanosoma brucei
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DOI：
10.1016/s0166-6851(96)02815-0
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发表时间：
1997-03-01
影响因子：
1.5
通讯作者：
Clayton, C
中科院分区：
文献类型：
--
作者：
Biebinger, S;Wirtz, LE;Clayton, C
我们先前描述了一种在布氏锥虫前循环型中对外源基因表达进行控制的系统，该系统依赖于一种四环素诱导的阻遏物与位于多聚（ADP - 核糖）聚合酶（PARP）启动子转录起始位点的操纵序列的结合。重组构建体被引入核糖体RNA重复序列的非转录间隔区，其方向与rRNA转录方向相反。利用该系统，基因表达可在四个数量级上进行调控，但无法表达毒性基因产物，因为重组锥虫的筛选依赖于诱导型启动子的活性。在此我们描述了包含两个启动子的载体的特性：一个是四环素诱导型启动子，用于驱动毒性产物的表达；另一个是组成型启动子，用于驱动选择标记的转录。在一些锥虫克隆中观察到相对较高水平的非诱导（非四环素依赖）表达；这通常不是由于多个串联整合的质粒的通读或操纵序列突变所致。对多种在抗性标记、3' - 非翻译区（3' - UTR）和组成型启动子性质方面不同的构建体进行了测试。获得了能够在两个生活周期阶段成功表达毒性基因及其他基因且调控因子高达700倍的载体。（C）1997年，爱思唯尔科学出版社（Elsevier Science B.V.）
We previously described a system for exogenous control of gene expression in procyclic trypanosomes which depends upon the binding of a tetracycline-inducible repressor to operators situated at the transcriptional start site of the PARP promoter. The recombinant constructs are introduced into non-transcribed spacers of the ribosomal RNA repeat, in an orientation opposite to that of rRNA transcription. Using this system, gene expression could be regulated over four orders of magnitude, but it was not possible to express toxic gene products because selection of recombinant trypanosomes depended on the activity of the inducible promoter. We describe here the characteristics of vectors that include two promoters: a tetracycline-inducible one to drive expression of the toxic product, and a constitutive one to drive transcription of the selectable marker. Relatively high levels of non-induced (non-tetracycline-dependent) expression were seen in some trypanosome clones; this was not usually due to read-through of multiple tandemly-integrated plasmids or let operator mutations. A variety of constructs differing in resistance marker, 3'-untranslated region (3'-UTR) and the nature of the constitutive promoter was tested. Vectors allowing the successful expression of toxic and other genes in both life cycle stages with regulation factors of up to 700 fold were obtained. (C) 1997 Elsevier Science B.V.