Kim Hee-Sook, Li Zhen, Boothroyd Catharine, Cross George A M
Laboratory of Molecular Parasitology, The Rockefeller University, New York, NY 10065, USA; Laboratory of Lymphocyte Biology, The Rockefeller University, New York, NY 10065, USA.
Mol Biochem Parasitol. 2013 Sep;191(1):16-9. doi: 10.1016/j.molbiopara.2013.08.001. Epub 2013 Aug 13.
We describe two gene-knockout (KO) strategies in Trypanosoma brucei using Cre recombinase and loxP sites. Due to the limited number of selection markers for T. brucei, it has been difficult to generate a mutant with two genes knocked out and impractical to simultaneously knockout more than two genes, deterring detailed studies of important cellular mechanisms. The first KO strategy described can overcome the marker problem by allowing continuous re-use of drug-resistance markers. The same KO vector can be used to make a conditional KO system, when a gene of interest is essential for cell viability. As a gene of interest is removed from its original chromosomal locus by the induction of Cre recombinase, deletion is complete and instantaneous. This makes it easier to identify primary effects rather than having secondary effects obscuring phenotypic assessment, as is often the case with RNAi silencing.
我们描述了在布氏锥虫中使用Cre重组酶和loxP位点的两种基因敲除（KO）策略。由于布氏锥虫的选择标记数量有限，很难产生两个基因被敲除的突变体，同时敲除两个以上基因也不切实际，这阻碍了对重要细胞机制的详细研究。所描述的第一种KO策略可以通过允许连续重复使用耐药标记来克服标记问题。当感兴趣的基因对细胞活力至关重要时，相同的KO载体可用于构建条件性KO系统。由于通过Cre重组酶的诱导将感兴趣的基因从其原始染色体位点移除，缺失是完全且瞬时的。这使得更容易识别主要效应，而不是像RNAi沉默那样经常出现的由次要效应掩盖表型评估的情况。
