Toxoplasma gondii and the related Apicomplexan protozoan pathogens, Plasmodium, Cryptosporidium, and Eimeria, are obligate intracellular parasites which cause severe disease in their hosts. The recent development of transient transfection of Toxoplasma permits the development of strategies utilizing "reverse genetics" to identify molecules critical to parasite survival within the host. We have utilized transfection of Toxoplasma tachyzoites to stably complement a sag1 (or p30) mutant that does not make detectable SAG1. Transfection of mutants with the wild-type SAG1 gene resulted in transient expression of SAG1 in approximately 15-20% of the transfected population. Stable transformants were enriched by repeated sorting of live parasites using a fluorescein-labeled monoclonal antibody specific for SAG1. Cloned recombinant parasites expressed SAG1 at wild-type levels and maintained expression for over 5 months after transfection (approximately 300 divisions). Cloned transformants (which proved to be siblings) carried both the mutated gene and one copy of the transfected gene which had inserted randomly into the Toxoplasma genome.
