Improved techniques for endogenous epitope tagging and gene deletion in Toxoplasma gondii

Rajendra Upadhya, Kami Kim, Ruth Hogue-Angeletti, Louis M. Weiss

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

Toxoplasma gondii is an excellent model organism for studies on the biology of the Apicomplexa due to its ease of in vitro cultivation and genetic manipulation. Large-scale reverse genetic studies in T. gondii have, however, been difficult due to the low frequency of homologous recombination. Efforts to ensure homologous recombination have necessitated engineering long flanking regions in the targeting construct. This requirement makes it difficult to engineer chromosomally targeted epitope tags or gene knock out constructs only by restriction enzyme mediated cloning steps. To address this issue we employed multisite Gateway® recombination techniques to generate chromosomal gene manipulation targeting constructs. Incorporation of 1.5 to 2.0. kb flanking homologous sequences in PCR generated targeting constructs resulted in 90% homologous recombination events in wild type T. gondii (RH strain) as determined by epitope tagging and target gene deletion experiments. Furthermore, we report that split marker constructs were equally efficient for targeted gene disruptions using the T. gondii UPRT gene locus as a test case. The methods described in this paper represent an improved strategy for efficient epitope tagging and gene disruptions in T. gondii.

Original languageEnglish (US)
Pages (from-to)103-113
Number of pages11
JournalJournal of Microbiological Methods
Volume85
Issue number2
DOIs
StatePublished - May 2011

Keywords

  • Epitope tagging
  • Gateway vectors
  • Gene deletion
  • PCR product mediated transfection
  • Toxoplasma gondii
  • UPRT knock out

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology
  • Microbiology (medical)

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