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

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

Research output: Contribution to journalArticle

20 Citations (Scopus)

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 2011

Fingerprint

Gene Targeting
Leishmania donovani
Inosine Monophosphate
Genes
Ligation
Oxidoreductases
Leishmania
DNA Restriction Enzymes
Drug Resistance

Keywords

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

ASJC Scopus subject areas

  • Molecular Biology
  • Parasitology

Cite this

A rapid, efficient and economical method for generating leishmanial gene targeting constructs. / Fulwiler, Audrey L.; Soysa, D. Radika; Ullman, Buddy; Yates, Phillip.

In: Molecular and Biochemical Parasitology, Vol. 175, No. 2, 02.2011, p. 209-212.

Research output: Contribution to journalArticle

Fulwiler, Audrey L. ; Soysa, D. Radika ; Ullman, Buddy ; Yates, Phillip. / A rapid, efficient and economical method for generating leishmanial gene targeting constructs. In: Molecular and Biochemical Parasitology. 2011 ; Vol. 175, No. 2. pp. 209-212.
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