Adaptation of signature-tagged mutagenesis for Toxoplasma gondii: a negative screening strategy to isolate genes that are essential in restrictive growth conditions.
The obligate intracellular parasite Toxoplasma gondii can infect virtually any nucleated cell in any warm-blooded host. Through the effort of many researchers, we are beginning to learn what makes T. gondii such a successful protozoan parasite. A high throughput genetic screen that allows simultaneous examination of a large panel of mutants would greatly facilitate a global investigation of this parasite. Signature-tagged mutagenesis uses a unique DNA sequence to tag an individual mutant so that it can later be identified within a pool. This system allows the efficient identification of parasites carrying mutations in genes that are essential for growth in restrictive but not permissive conditions. We have generated a bank of approximately 4900 signature-tagged T. gondii tachyzoites represented in 89 pools, each of which contains 60 uniquely tagged mutant parasites. We have demonstrated the usefulness of this negative screening strategy with a tissue culture model for pyrimidine salvage using resistance to the pro-drug FUDR. Mutants that are defective for growth in any defined growth condition versus standard tissue culture conditions can now be identified (eg, sensitive to a specific drug, growth in a specialized cell line, or growth within animals).
Knoll
L J
LJ
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305-5124, USA.
Furie
G L
GL
Boothroyd
J C
JC
D000818:Animals; D017125:Genes, Protozoan; D016296:Mutagenesis / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000379:methods; D012641:Selection, Genetic; D014122:Toxoplasma / Q000235:genetics*