CRE recombinase-based positive-negative selection systems for genetic manipulation in Trypanosoma brucei

Michael D. Scahill, Irena Pastar, George A.M. Cross

Research output: Contribution to journalArticlepeer-review

28 Scopus citations

Abstract

The limited repertoire of drug-resistance markers imposes a serious obstacle to genetic manipulation of Trypanosoma brucei. Here we describe experiments with a fusion protein that allows positive selection for genome integration followed by CRE recombinase-mediated excision of the marker cassette that can be selected by ganciclovir, although the excision event is so efficient that selection is not strictly necessary. We describe two variants of the tetracycline-inducible pLEW100-based CRE-expression vector that reduced its toxicity when stably integrated into the genome, and we demonstrate that transient transfection of circular pLEW100-CRE is highly efficient at catalyzing marker excision. We used this approach to delete the last two enzymes of the pyrimidine synthesis pathway, creating a cell line that is resistant to fluoroorotic acid, which would allow the same enzymes (PYR6-5) to be used as an alternative negative selectable marker.

Original languageEnglish (US)
Pages (from-to)73-82
Number of pages10
JournalMolecular and Biochemical Parasitology
Volume157
Issue number1
DOIs
StatePublished - Jan 2008

Keywords

  • CRE recombinase
  • Culture
  • Fluoroorotic acid
  • Ganciclovir
  • Genetic tools
  • Negative selection
  • Pyrimidine auxotroph
  • Pyrimidine synthesis
  • Thymidine
  • Trypanosoma brucei
  • ura3

ASJC Scopus subject areas

  • Parasitology
  • Molecular Biology

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