Abstract
BackgroundMuch of the Plasmodium falciparum genome encodes hypothetical proteins with limited homology to other organisms. A lack of robust tools for genetic manipulation of the parasite limits functional analysis of these hypothetical proteins and other aspects of the Plasmodium genome. Transposon mutagenesis has been used widely to identify gene functions in many organisms and would be extremely valuable for functional analysis of the Plasmodium genome.ResultsIn this study, we investigated the lepidopteran transposon, piggyBac, as a molecular genetic tool for functional characterization of the Plasmodium falciparum genome. Through multiple transfections, we generated 177 unique P. falciparum mutant clones with mostly single piggyBac insertions in their genomes. Analysis of piggyBac insertion sites revealed random insertions into the P. falciparum genome, in regards to gene expression in parasite life cycle stages and functional categories. We further explored the possibility of forward genetic studies in P. falciparum with a phenotypic screen for attenuated growth, which identified several parasite genes and pathways critical for intra-erythrocytic development.ConclusionOur results clearly demonstrate that piggyBac is a novel, indispensable tool for forward functional genomics in P. falciparum that will help better understand parasite biology and accelerate drug and vaccine development.
Highlights
Much of the Plasmodium falciparum genome encodes hypothetical proteins with limited homology to other organisms
As piggyBac transposase is the functional enzyme catalyzing the integration event, we hypothesized that increased expression of the transposase with a stronger promoter would result in increased transformation efficiency
By using the piggyBac transposable element in P. falciparum, we have clearly demonstrated the pLBacII-HDH-KanOri- The kanamycin resistance gene and pUC origin of replication were amplified as a single fragment by PCR from the vector pEGFP-C1 (Clontech) using primers F-ATGATGATGGGATCCAAATGTGCGCGGAACCCC and R-ATGATGATGGGATCCGCAAAAGGCCAGCAAAAGG and cloned into pGEM-Teasy vector (Promega)
Summary
Much of the Plasmodium falciparum genome encodes hypothetical proteins with limited homology to other organisms. A lack of robust tools for genetic manipulation of the parasite limits functional analysis of these hypothetical proteins and other aspects of the Plasmodium genome. A continuous rise in parasite drug-resistance has further hindered malaria control strategies and resulted in increased number of deaths in the last few years [2]. BMC Microbiology 2009, 9:83 http://www.biomedcentral.com/1471-2180/9/83 biology [3,4,5,6,7,8] Despite these enormous efforts, Plasmodium genomes continue to be perplexing with more than 50% of the genes coding for hypothetical proteins with limited homology to model organisms. High throughput methods for identification of gene functions are imperative to better understand parasite biology and develop effective disease control strategies. Generating gene disruptions through classic reverse genetic approaches is a complex and inefficient process in P. falciparum, due to an extremely low parasite transfection efficiency and the parasite's ability to maintain transfected plasmids as episomes, resulting in only less than 1% of the total annotated genes knocked out far [9,10]
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