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.

Plasmodium berghei and Plasmodium chabaudi are widely used model malaria species. Comparison of their genomes, integrated with proteomic and microarray data, with the genomes of Plasmodium falciparum and Plasmodium yoelii revealed a conserved core of 4500 Plasmodium genes in the central regions of the 14 chromosomes and highlighted genes evolving rapidly because of stage-specific selective pressures. Four strategies for gene expression are apparent during the parasites' life cycle: (i) housekeeping; (ii) host-related; (iii) strategy-specific related to invasion, asexual replication, and sexual development; and (iv) stage-specific. We observed posttranscriptional gene silencing through translational repression of messenger RNA during sexual development, and a 47-base 3′ untranslated region motif is implicated in this process.

Anopheles mosquitoes are major vectors of human malaria in Africa. Large variation exists in the ability of mosquitoes to serve as vectors and to transmit malaria parasites, but the molecular mechanisms that determine vectorial capacity remain poorly understood. We report that the hemocyte-specific complement-like protein TEP1 from the mosquito Anopheles gambiae binds to and mediates killing of midgut stages of the rodent malaria parasite Plasmodium berghei. The dsRNA knockdown of TEP1 in adults completely abolishes melanotic refractoriness in a genetically selected refractory strain. Moreover, in susceptible mosquitoes this knockdown increases the number of developing parasites. Our results suggest that the TEP1-dependent parasite killing is followed by a TEP1-independent clearance of dead parasites by lysis and/or melanization. Further elucidation of the molecular mechanisms of TEP1-mediated parasite killing will be of great importance for our understanding of the principles of vectorial capacity in insects.

Translational repression (TR) of mRNAs plays an important role in sexual differentiation and gametogenesis in multicellular eukaryotes. We show here that TR and mRNA turnover are key influences on stage specific gene expression in the protozoan Plasmodium. The DDX6 class RNA helicase, DOZI (development of zygote inhibited), is found in cytoplasmic bodies of female, blood stage gametocytes in a complex with mRNA species known to be translationally repressed. Genetic disruption of pbdozi inhibits the formation of translationally quiescent mRNPs and targets these and other (>350 different) transcripts to the degradation pathway rather than directing them to translating polysomes, preventing zygote development prior to meiosis in the mosquito. Thus TR is essential in malaria parasite development. A full catalogue of the proteins and processes associated with DOZI (the first described malaria parasite TR-effector protein) might lead to novel approaches to prevent parasite development.Translational repression (TR) of mRNAs in higher eukaryotes controls temporal expression of specific protein cascades or directs the location of translation within a cell, and is prominent after gamete fertilisation (zygote formation) in the early embryo when de novo transcription of mRNA is restricted (1-5). The hallmark of repression is the assembly of certain mRNAs together with proteins into quiescent messenger ribonucleoprotein particles (mRNPs) where these transcripts are stored for translation at a later time. The DDX6-family of DEAD-box RNA helicases is tightly linked both to storage of mRNAs encoding proteins associated with progression through meiosis into translationally silent mRNPs and with the * To whom correspondence should be addressed waters@lumc.nl. Post-fertilsation development of Plasmodium zygotes in the mosquito relys on the formation of translationally quiescent mRNPs in blood-stage, female sexual precursor cells Supporting Online Material www.sceincemag.org Materials and Methods Figs. S1 to S4 Tables S1 to S4 References

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