Tagging genes and trapping promoters in Toxoplasma gondii by insertional mutagenesis

David S. Roos, William Sullivan, Boris Striepen, Wolfgang Bohne, Robert G K Donald

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

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 parsites. 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.

Original languageEnglish (US)
Pages (from-to)112-122
Number of pages11
JournalMethods in Enzymology
Volume13
Issue number2
DOIs
StatePublished - Oct 1997
Externally publishedYes

Fingerprint

Mutagenesis
Insertional Mutagenesis
Toxoplasma
Thymidylate Synthase
Parasites
Genes
Oxidoreductases
Genome
Pyrimethamine
Electroporation
Haploidy
Homologous Recombination
DNA
Gene Silencing
Introns
Genetic Recombination
Drosophila
Plasmids
Complementary DNA
Yeasts

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Tagging genes and trapping promoters in Toxoplasma gondii by insertional mutagenesis. / Roos, David S.; Sullivan, William; Striepen, Boris; Bohne, Wolfgang; Donald, Robert G K.

In: Methods in Enzymology, Vol. 13, No. 2, 10.1997, p. 112-122.

Research output: Contribution to journalArticle

Roos, David S. ; Sullivan, William ; Striepen, Boris ; Bohne, Wolfgang ; Donald, Robert G K. / Tagging genes and trapping promoters in Toxoplasma gondii by insertional mutagenesis. In: Methods in Enzymology. 1997 ; Vol. 13, No. 2. pp. 112-122.
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