A novel 'gene insertion/marker out' (GIMO) method for transgene expression and gene complementation in rodent malaria parasites.
A novel 'gene insertion/marker out' (GIMO) method for transgene expression and gene complementation in rodent malaria parasites.
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DOI：
10.1371/journal.pone.0029289
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Khan SM
中科院分区：
文献类型：
--
作者：
Lin JW;Annoura T;Sajid M;Chevalley-Maurel S;Ramesar J;Klop O;Franke-Fayard BM;Janse CJ;Khan SM
疟疾寄生虫生物学研究极大地受益于反向遗传技术的应用，特别是通过分析基因缺失突变体和研究表达异源或突变蛋白质的转基因寄生虫。然而，疟原虫中的转染受到药物选择性标记缺乏的限制，这阻碍了相同突变体的后续遗传修饰。我们报告了一种新的“基因插入/标记出”（GIMO）的方法，两个啮齿类疟原虫，它使用负选择，以快速生成转基因突变体准备后续修改的发展。我们已经创建了伯氏疟原虫ANKA和约氏疟原虫17 XNL的参考母系，其用作GIMO转染的受体寄生虫。与现有的方案相比，GIMO转染大大简化和加速了表达异源蛋白的突变体的产生，不含耐药基因，并且需要少得多的实验室动物。此外，我们证明GIMO转染也是一种简单而快速的方法，用于基因缺失或突变的突变体的遗传互补。GIMO转染程序的实施将大大加强疟原虫反向遗传研究。
Research on the biology of malaria parasites has greatly benefited from the application of reverse genetic technologies, in particular through the analysis of gene deletion mutants and studies on transgenic parasites that express heterologous or mutated proteins. However, transfection in Plasmodium is limited by the paucity of drug-selectable markers that hampers subsequent genetic modification of the same mutant. We report the development of a novel ‘gene insertion/marker out’ (GIMO) method for two rodent malaria parasites, which uses negative selection to rapidly generate transgenic mutants ready for subsequent modifications. We have created reference mother lines for both P. berghei ANKA and P. yoelii 17XNL that serve as recipient parasites for GIMO-transfection. Compared to existing protocols GIMO-transfection greatly simplifies and speeds up the generation of mutants expressing heterologous proteins, free of drug-resistance genes, and requires far fewer laboratory animals. In addition we demonstrate that GIMO-transfection is also a simple and fast method for genetic complementation of mutants with a gene deletion or mutation. The implementation of GIMO-transfection procedures should greatly enhance Plasmodium reverse-genetic research.