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Mol Biochem Parasitol. 2011 Feb;175(2):209-12. doi: 10.1016/j.molbiopara.2010.10.008. Epub 2010 Nov 3.

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

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  • 1Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR 97239-3098, USA.

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.

Copyright © 2010 Elsevier B.V. All rights reserved.

PMID:
21055426
[PubMed - indexed for MEDLINE]
PMCID:
PMC3018707
Free PMC Article

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