Biebinger S, Wirtz L E, Lorenz P, Clayton C
Zentrum für Molekular Biologie, Heidelberg, Germany.
Mol Biochem Parasitol. 1997 Mar;85(1):99-112. doi: 10.1016/s0166-6851(96)02815-0.
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 products, 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 tet 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.
我们之前描述了一种用于前循环型锥虫基因表达外源控制的系统，该系统依赖于四环素诱导型阻遏物与位于PARP启动子转录起始位点的操纵子的结合。重组构建体被导入核糖体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节，但由于重组锥虫的选择依赖于诱导型启动子的活性，因此无法表达有毒基因产物。我们在此描述了包含两个启动子的载体的特性：一个四环素诱导型启动子用于驱动有毒产物的表达，一个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（非四环素依赖性）表达；这通常不是由于多个串联整合质粒的通读或四环素操纵子突变所致。测试了多种在抗性标记、3'非翻译区（3'-UTR）和组成型启动子性质方面不同的构建体。获得了能够在两个生命周期阶段成功表达有毒和其他基因且调节因子高达700倍的载体。
