Santos Renato E R S, Silva Gabriel L A, Santos Elaine V, Duncan Samuel M, Mottram Jeremy C, Damasceno Jeziel D, Tosi Luiz R O
Department of Cell and Molecular Biology, Ribeirão Preto Medical School, University of São Paulo; Ribeirão Preto, SP, Brazil.
Wellcome Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, United Kingdom.
Mol Biochem Parasitol. 2017 Sep;216:45-48. doi: 10.1016/j.molbiopara.2017.06.006. Epub 2017 Jun 16.
Here we present the establishment of an inducible system based on the dimerizable Cre recombinase (DiCre) for controlled gene expression in the protozoan parasite Leishmania. Rapamycin-induced DiCre activation promoted efficient flipping and expression of gene products in a time and dose-dependent manner. The DiCre flipping activity induced the expression of target genes from both integrated and episomal contexts broadening the applicability of the system. We validated the system by inducing the expression of both full length and truncated forms of the checkpoint protein Rad9, which revealed that the highly divergent C-terminal domain of Rad9 is necessary for proper subcellular localization. Thus, by establishing the DiCre-based inducible system we have created and validated a robust new tool for assessing gene function in Leishmania.
在此，我们展示了基于二聚化Cre重组酶（DiCre）建立的诱导系统，用于原生动物寄生虫利什曼原虫中基因的可控表达。雷帕霉素诱导的DiCre激活以时间和剂量依赖的方式促进了基因产物的有效翻转和表达。DiCre翻转活性诱导了整合型和游离型背景下靶基因的表达，拓宽了该系统的适用性。我们通过诱导检查点蛋白Rad9全长和截短形式的表达来验证该系统，结果表明Rad9高度不同的C末端结构域对于正确的亚细胞定位是必需的。因此，通过建立基于DiCre的诱导系统，我们创建并验证了一种用于评估利什曼原虫基因功能的强大新工具。
