CRE recombinase-based positive-negative selection systems for genetic manipulation in Trypanosoma brucei
CRE recombinase-based positive-negative selection systems for genetic manipulation in Trypanosoma brucei
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DOI：
10.1016/j.molbiopara.2007.10.003
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发表时间：
2008-01-01
影响因子：
1.5
通讯作者：
Cross, George A. M.
中科院分区：
文献类型：
--
作者：
Scahill, Michael D.;Pastar, Irena;Cross, George A. M.
有限的耐药标记库给布氏锥虫的遗传操作带来了严重的障碍。在这里，我们描述了使用融合蛋白的实验，该融合蛋白允许对基因组整合进行正选择，随后是Cre重组酶介导的标记盒切除，该标记盒可以被更昔洛韦选择，尽管切除事件是如此有效，选择并不是严格必要的。我们描述了四环素诱导的基于Plew 100的Cre表达载体的两种变体，当稳定整合到基因组中时，它们降低了其毒性，我们证明了瞬时转染环Plew 100-Cre在催化标记切除方面是高效的。我们使用这种方法删除了嘧啶合成途径的最后两个酶，创建了一个对氟罗丹酸具有抗性的细胞系，这将允许将相同的酶(PYR6-5)用作替代的阴性选择标记。(C)2007年，爱思唯尔出版。
The limited repertoire of drug-resistance markers imposes a serious obstacle to genetic manipulation of Trypanosoma brucei. Here we describe experiments with a fusion protein that allows positive selection for genome integration followed by CRE recombinase-mediated excision of the marker cassette that can be selected by ganciclovir, although the excision event is so efficient that selection is not strictly necessary. We describe two variants of the tetracycline-inducible pLEW 100-based CRE-expression vector that reduced its toxicity when stably integrated into the genome, and we demonstrate that transient transfection of circular pLEW 100-CRE is highly efficient at catalyzing marker excision. We used this approach to delete the last two enzymes of the pyrimidine synthesis pathway, creating a cell line that is resistant to fluoroorotic acid, which would allow the same enzymes (PYR6-5) to be used as an alternative negative selectable marker. (c) 2007 Published by Elsevier B.V.