Gene Targeting in Malaria Parasites

Gene targeting, which permits alteration of a chosen gene in a predetermined way by homologous recombination, is an emerging technology in malaria research. Soon after the development of techniques for stable transformation of red blood cell stages ofPlasmodium falciparumandPlasmodium berghei,genes of interest were disrupted in the two species. The main limitations of gene targeting in malaria parasites result from the intracellular growth and slow replication of these parasites. On the other hand, the technology is facilitated by the very high rate of homologous recombination following transformation with targeting constructs (∼100%). Here, we describe (i) the vector design and the type of mutation that may be generated in a target locus, (ii) the selection and screening strategies that can be used to identify clones with the desired modification, and (iii) the protocol that was used for disrupting the circumsporozoite protein (CS) and thrombospondin-related anonymous protein (TRAP) genes ofP. berghei.