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Mol Biochem Parasitol. 2013 Sep;191(1):16-9. doi: 10.1016/j.molbiopara.2013.08.001. Epub 2013 Aug 13.

Strategies to construct null and conditional null Trypanosoma brucei mutants using Cre-recombinase and loxP.

Author information

1
Laboratory of Molecular Parasitology, The Rockefeller University, New York, NY 10065, USA; Laboratory of Lymphocyte Biology, The Rockefeller University, New York, NY 10065, USA. Electronic address: khee-sook@rockefeller.edu.

Abstract

We describe two gene-knockout (KO) strategies in Trypanosoma brucei using Cre recombinase and loxP sites. Due to the limited number of selection markers for T. brucei, it has been difficult to generate a mutant with two genes knocked out and impractical to simultaneously knockout more than two genes, deterring detailed studies of important cellular mechanisms. The first KO strategy described can overcome the marker problem by allowing continuous re-use of drug-resistance markers. The same KO vector can be used to make a conditional KO system, when a gene of interest is essential for cell viability. As a gene of interest is removed from its original chromosomal locus by the induction of Cre recombinase, deletion is complete and instantaneous. This makes it easier to identify primary effects rather than having secondary effects obscuring phenotypic assessment, as is often the case with RNAi silencing.

KEYWORDS:

ALD; BLE; BSD; Conditional gene knockout; Cre-recombinase; GCV; Gene knockout; HSVTK or TK; HYG; Herpes simplex virus thymidine kinase; KO; NEO; ORF; PUR; SAS; TUB; Trypanosoma brucei; UTR; aldolase; blasticidin-resistance gene; bleomycin-resistance gene; gancyclovir; hygromycin-resistance gene; knockout; neomycin-resistance gene; open reading frame; puromycin-resistance gene; splice accepter site; tubulin; untranslated region

PMID:
23954366
PMCID:
PMC3830529
DOI:
10.1016/j.molbiopara.2013.08.001
[Indexed for MEDLINE]
Free PMC Article

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