Improved techniques for endogenous epitope tagging and gene deletion in Toxoplasma gondii

Toxoplasma gondii is an excellent model organism for studies on the biology of the Apicomplexa due to its ease of in vitro cultivation and genetic manipulation. Large-scale reverse genetic studies in T. gondii have, however, been difficult due to the low frequency of homologous recombination. Effort...

Full description

Saved in:
Bibliographic Details
Published in:Journal of microbiological methods 2011-05, Vol.85 (2), p.103-113
Main Authors: Upadhya, Rajendra, Kim, Kami, Hogue-Angeletti, Ruth, Weiss, Louis M.
Format: Article
Language:English
Subjects:
Apicomplexa
Biological and medical sciences
Cell Line
engineering
Epitope tagging
Fundamental and applied biological sciences. Psychology
Gateway vectors
Gene Deletion
Gene Targeting - methods
genes
genetic engineering
homologous recombination
Humans
loci
Microbiology
Mutagenesis, Insertional - methods
PCR product mediated transfection
polymerase chain reaction
protein tagging
Protozoan Proteins - genetics
restriction endonucleases
sequence homology
Toxoplasma - genetics
Toxoplasma gondii
UPRT knock out
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Toxoplasma gondii is an excellent model organism for studies on the biology of the Apicomplexa due to its ease of in vitro cultivation and genetic manipulation. Large-scale reverse genetic studies in T. gondii have, however, been difficult due to the low frequency of homologous recombination. Efforts to ensure homologous recombination have necessitated engineering long flanking regions in the targeting construct. This requirement makes it difficult to engineer chromosomally targeted epitope tags or gene knock out constructs only by restriction enzyme mediated cloning steps. To address this issue we employed multisite Gateway® recombination techniques to generate chromosomal gene manipulation targeting constructs. Incorporation of 1.5 to 2.0 kb flanking homologous sequences in PCR generated targeting constructs resulted in 90% homologous recombination events in wild type T. gondii (RH strain) as determined by epitope tagging and target gene deletion experiments. Furthermore, we report that split marker constructs were equally efficient for targeted gene disruptions using the T. gondii UPRT gene locus as a test case. The methods described in this paper represent an improved strategy for efficient epitope tagging and gene disruptions in T. gondii.
ISSN:0167-7012
1872-8359
DOI:10.1016/j.mimet.2011.02.001