The molecular genetics core is intended to provide technical support to the various laboratories involved in this cooperative project, facilitating application of the powerful new tools developed during the first funding cycle. To summarize existing technology available for Toxoplasma, it is now possible to express recombinant molecules both transiently and as stable transformants, as episomes or integrated into the genome, and as single copy or multicopy transgenes. Stable integration can arise either by random nonhomologous recombination, single site homologous recombination (producing a duplication at the transfected locus) or as a perfect allelic replacement (or gene knockout). Many of these aspects can be controlled by the use of appropriate vectors, selectable markers, and transformation conditions. In sum, it is now possible to apply virtually the entire spectrum of molecular genetic tools to Toxoplasma. The extraordinarily high frequency of stable transformation observed (up to 6% of viable transfected tachyzoites) permits a variety of important applications. The feasibility of gene cloning by complementation may allow identification of drug resistance loci for parasiticidal agents whose mechanism of action is unknown(Roos). Insertional mutagenesis has already been used to tag the adenosine kinase, HXGPRT, and UPRT loci (Joiner, Ullman), leading to the successful cloning of HXGPRT and UPRT cDNAs (AK should follow shortly). These techniques also provide an alternative to complementation for the identification of unknown targets (Roos). Gene knock-outs supply a direct means to validate promising targets as essential for parasite survival (Joiner, Ullman), and allelic replacement permits critical assessment of molecular models in transgenic parasites (Ullman). Efforts will also be directed toward the development of further molecular tools (better - and safer - selectable markers, 'hit-and- run' mutagenesis vectors, episomal libraries and shuttle vectors) and reagents (including stage-specific clones and libraries for different parasite strains). Finally, because these techniques bring unprecedented power to the investigation of T. gondii, in keeping with the targeted 'National Cooperative' nature of the NCDDG research, we will continue to assist in technology transfer and collaboration with other groups around the world, when such cooperation is likely to further the ultimate goal of improved treatment options to eliminate or alleviate the burden of AIDS toxoplasmosis.
