A rapid, efficient and economical method for generating leishmanial gene targeting constructs

Audrey L. Fulwiler, D. Radika Soysa, Buddy Ullman, Phillip A. Yates

Research output: Contribution to journalArticle

21 Scopus citations

Abstract

Targeted gene replacement is a powerful tool in Leishmania genetics that can be time-consuming to implement. One tedious aspect that delays progress is the multi-step construction of gene targeting vectors. To accelerate this process, we developed a streamlined method that allows the assembly of a complete targeting vector from all its constituent parts in a single-step multi-fragment ligation. The individual components to be assembled are flanked by sites for the restriction endonuclease SfiI that generates non-identical, non-palindromic three base 3′-overhangs designed to allow annealing and ligation of the parts only in the proper order. The method was optimized by generating constructs for targeting the Leishmania donovani inosine monophosphate dehydrogenase gene (LdIMPDH) encoding six different drug resistance markers, and was found to be rapid and efficient. These constructs were successfully employed to generate heterozygous LdIMPDH gene replacement mutants. This method is adaptable for generating targeting vectors for a variety of species.

Original languageEnglish (US)
Pages (from-to)209-212
Number of pages4
JournalMolecular and Biochemical Parasitology
Volume175
Issue number2
DOIs
StatePublished - Feb 1 2011

Keywords

  • Gene knockout
  • Gene targeting
  • Homologous recombination
  • Leishmania
  • Multi-fragment ligation
  • Sfil restriction endonuclease

ASJC Scopus subject areas

  • Parasitology
  • Molecular Biology

Fingerprint Dive into the research topics of 'A rapid, efficient and economical method for generating leishmanial gene targeting constructs'. Together they form a unique fingerprint.

  • Cite this