Functional Analysis of Large Plasmodium falciparum genes

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DESCRIPTION (provided by applicant): Progress in malaria research has been substantially accelerated in recent years by genome sequencing of P. falciparum and other Plasmodium species. Application of systems biology tools to the genome data has identified new pathways and targets for drug and vaccine development. A critical component of confirming the potential of such targets is analyzing the function of the relevant gene(s) and determining the effect of interference with that function. P. falciparum is not amenable to traditional genetic approaches so function is tested by the introduction of directed mutations by transfection with plasmid constructs. There have been major strides in development of transfection methods for P. falciparum, hand-in-hand with the genome sequencing effort, and many critical insights have resulted. However, as yet there are no methods that allow deletion of internal sequences without gene disruption, complementation, allelic replacement, or mutagenesis for the central regions of long genes, like var genes. The var gene family encodes PfEMP1 proteins, which mediate cytoadherence and antigenic variation. Current methods for analysis of PfEMP1 adhesive properties rely on testing isolated domains and the data may not be representative of function when the domain is part of a large protein. The importance of PfEMP1 to malaria pathogenesis makes it critical to devise methods for genetic analysis of central regions of long genes. We here propose a method to integrate sequences into central regions of long P. falciparum genes without ablation of expression, allowing detailed analysis of gene function for critically important genes.