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

The first KO strategy allows continuous reuse of selection markers for double or triple mutant construction and the second allows the molecular functions of essential genes to be investigated by conditional and instantaneous gene deletion. •Cre-loxP knockout (KO) system allows continuous reuse of dr...

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Bibliographic Details
Published in:Molecular and biochemical parasitology 2013-09, Vol.191 (1), p.16-19
Main Authors: Kim, Hee-Sook, Li, Zhen, Boothroyd, Catharine, Cross, George A.M.
Format: Article
Language:English
Subjects:
cell viability
Conditional gene knockout
Cre-recombinase
drug resistance
Gene knockout
Gene Knockout Techniques - methods
genes
Genes, Essential
Genetic Vectors
Genetics, Microbial - methods
Integrases - metabolism
loci
Molecular Biology - methods
mutants
parasitology
Parasitology - methods
RNA interference
Selection, Genetic
Trypanosoma brucei
Trypanosoma brucei brucei - genetics
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Summary:The first KO strategy allows continuous reuse of selection markers for double or triple mutant construction and the second allows the molecular functions of essential genes to be investigated by conditional and instantaneous gene deletion. •Cre-loxP knockout (KO) system allows continuous reuse of drug selection markers.•Cre-loxP KO can be used to study genetic interaction between multiple pathways.•Conditional KO (cKO) using Cre-loxP is useful to study essential genes. 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.
ISSN:0166-6851
1872-9428
DOI:10.1016/j.molbiopara.2013.08.001