基于双 Cre 系统的利什曼原虫基因诱导性破坏

DiCre-Based Inducible Disruption of Leishmania Genes.

作者信息

Duncan Samuel M, Myburgh Elmarie, Alves-Ferreira Eliza V, Mottram Jeremy C

机构信息

Division of Biological Chemistry and Drug Discovery, Wellcome Trust Centre for Anti-infectives Research, University of Dundee, Dundee, UK.

Department of Biology, York Biomedical Research Institute, University of York, York, UK.

出版信息

Methods Mol Biol. 2019;1971:211-224. doi: 10.1007/978-1-4939-9210-2_10.

DOI:10.1007/978-1-4939-9210-2_10

PMID:30980305

Abstract

Conditional gene deletion using dimerizable Cre recombinase (DiCre) is so far the best developed system for the phenotypic analysis of essential genes in Leishmania species. Here, we describe a protocol for the generation of a conditional gene deletion mutant and the subsequent inducible deletion of a target gene. Leishmania parasites are genetically modified to express two inactive Cre subunits (DiCre) and a single LoxP-flanked version of a target gene in a context where both endogenous copies of the gene have been deleted. Treatment with rapamycin dimerizes the DiCre subunits, resulting in activation of the enzyme, recombination between the LoxP sites, and excision of the LoxP-flanked target gene. Subsequent phenotyping allows for the analysis of essential gene function.

摘要

迄今为止，使用可二聚化的Cre重组酶（DiCre）进行条件性基因缺失是用于利什曼原虫属中必需基因表型分析的最完善的系统。在此，我们描述了一种生成条件性基因缺失突变体以及随后诱导缺失靶基因的方案。利什曼原虫寄生虫经过基因改造，以在靶基因的两个内源性拷贝均已缺失的背景下表达两个无活性的Cre亚基（DiCre）和单个loxP侧翼的靶基因版本。用雷帕霉素处理可使DiCre亚基二聚化，从而导致酶激活、loxP位点之间的重组以及loxP侧翼靶基因的切除。随后的表型分析有助于对必需基因功能进行分析。