High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei

被引：470

作者：

Janse, Chris J. ^{[1
]}

Ramesar, Jai ^{[1
]}

Waters, Andrew P. ^{[1
]}

机构：

[1] Leiden Univ, Med Ctr, NL-2300 RC Leiden, Netherlands

来源：

NATURE PROTOCOLS | 2006年 / 1卷 / 01期

关键词：

D O I：

10.1038/nprot.2006.53

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

This protocol describes a method of genetic transformation for the rodent malaria parasite Plasmodium berghei with a high transfection efficiency of 10(-3) - 10(-4). It provides methods for: (i) in vitro cultivation and purification of the schizont stage; (ii) transfection of DNA constructs containing drug-selectable markers into schizonts using the nonviral Nucleofector technology; and (iii) injection of transfected parasites into mice and subsequent selection of mutants by drug treatment in vivo. Drug selection is described for two ( antimalarial) drugs, pyrimethamine and WR92210. The drug-selectable markers currently in use are the pyrimethamine-resistant dihydrofolate reductase (dhfr) gene of Plasmodium or Toxoplasma gondii and the DHFR gene of humans that confer resistance to pyrimethamine and WR92210, respectively. This protocol enables the generation of transformed parasites within 10 - 15 d. Genetic modification of P. berghei is widely used to investigate gene function in Plasmodium, and this protocol for high-efficiency transformation will enable the application of large-scale functional genomics approaches.

引用

页码：346 / 356

页数：11

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