Structure–Function Analysis of Malaria Proteins by Gene Targeting – A Response

Abstract

We have generated transgenic Plasmodium berghei parasites in which the endogenous gene encoding thrombospondin-related anonymous protein (TRAP) was replaced by either wild-type (WT) P. falciparum TRAP (PfTRAP) or by mutated versions of PfTRAP carrying amino acids substitutions or deletions in the A domain or in the thrombospondin-related motif1. This strategy was employed in a structure–function analysis, which aimed to circumvent limitations due to the availability of only one selectable marker to transform P. berghei. We did not attempt to modify the endogenous gene encoding TRAP by using an insertion-targeting approach because we were (and still are) convinced that genetic reversion is a limitation in structure–function studies. A reversion frequency of 1% should not be underestimated. If the revertant parasites were 50–100% more efficient than the mutated ones in infecting target cells or in gliding motility, their presence would confound the phenotypic analysis of the mutated parasites.