Tagging genes and trapping promoters in Toxoplasma gondii by insertional mutagenesis.
Plasmid vectors that incorporate sequence elements from the dehydrofolate reductase-thymidylate synthase (DHFR-TS) locus of Toxoplasma gondii integrate into the parasite genome with remarkably high frequency (>1% of transfected parasites). These vectors may-but need not-include mutant DHFR-TS alleles that confer pyrimethamine resistance to transgenic parasites. Large genomic constructs integrate at the endogenous locus by homologous recombination, but cDNA-derived sequences lacking long stretches of contiguous genomic DNA (due to intron excision) typically integrate into chromosomal DNA by nonhomologous recombination. Nonhomologous integration occurs effectively at random; and coupled with the high frequency of transformation, this allows a large fraction of the parasite genome to be tagged in a single electroporation cuvette. Genomic tagging permits insertional mutagenesis studies conceptually analogous to transposon mutagenesis in bacteria, yeast, Drosophila, etc. In theory (and, thus far, in practice), this allows identification of any gene whose inactivation is not lethal to the haploid tachyzoite form of T. gondii and for which a suitable selection or screen is available. Transformation vectors can be engineered to facilitate rescue of the tagged locus and to include a variety of reporters or selectable markers. Genetic strategies are also possible, using reporters whose function can be assayed by metabolic, visual, or immunological screens to "trap" genes that are activated (or inactivated) under various conditions of interest.
Roos
D S
DS
Department of Biology, University of Pennsylvania, 415 South University Avenue, Philadelphia, Pennsylvania, 19104-6018, USA. droos@sas.upenn.edu
Sullivan
W J
WJ
Striepen
B
B
Bohne
W
W
Donald
R G
RG
D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D018076:DNA, Complementary; D018274:Electroporation; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D007041:Hypoxanthine Phosphoribosyltransferase / Q000096:biosynthesis; D007438:Introns; D008164:Luminescent Proteins / Q000096:biosynthesis; D016254:Mutagenesis, Insertional / Q000379:methods*; D009173:Mycophenolic Acid / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011739:Pyrimethamine / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis; D013940:Thymidylate Synthase / Q000096:biosynthesis; D014122:Toxoplasma / Q000187:drug effects / Q000235:genetics*; D014162:Transfection / Q000379:methods*
D018076:DNA, Complementary; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D015500:Chloramphenicol O-Acetyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase; D009173:Mycophenolic Acid; D011739:Pyrimethamine