Chapter 3: Molecular Tools for Genetic Dissection of the Protozoan Parasite Toxoplasma gondii

Abstract

The genetic structure of Toxoplasma gondii is notable chiefly for being relatively conventional— similar to that of its mammalian host cells with respect to gene organization, codon usage, and nucleotide bias. These observations have led several investigators to examine the feasibility of molecular transformation in this parasite. This chapter outlines the use of several of the molecular genetic tools that have recently been developed for the T. gondii system. An introduction to parasite culture techniques is also provided. Recombinant molecules can be expressed either transiently or as stable transformants, as episomes or integrated into the parasite genome, and as single copy or multicopy transgenes. Stable integration can be produced by random nonhomologous recombination, single-site homologous recombination, or perfect gene replacement. Many of these outcomes can be selected specifically using appropriate vectors and transformation conditions. The extraordinarily high frequencies of stable transformation observed permit cloning by complementation, insertional mutagenesis/marker rescue, gene knock-outs, and allelic replacement. In combination with available classical and “cell-genetic” possibilities and physical and genetic mapping strategies, these tools provide a powerful arsenal for investigations into the biology of intracellular parasitism.