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Eukaryotic Cell, April 2009, p. 530-539, Vol. 8, No. 4
1535-9778/09/$08.00+0     doi:10.1128/EC.00358-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Tagging of Endogenous Genes in a Toxoplasma gondii Strain Lacking Ku80 {triangledown} ,{dagger}

My-Hang Huynh and Vern B. Carruthers*

Department of Microbiology and Immunology, University of Michigan School of Medicine, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109

Received 31 October 2008/ Accepted 2 February 2009

As with other organisms with a completed genome sequence, opportunities for performing large-scale studies, such as expression and localization, on Toxoplasma gondii are now much more feasible. We present a system for tagging genes endogenously with yellow fluorescent protein (YFP) in a {Delta}ku80 strain. Ku80 is involved in DNA strand repair and nonhomologous DNA end joining; previous studies in other organisms have shown that in its absence, random integration is eliminated, allowing the insertion of constructs with homologous sequences into the proper loci. We generated a vector consisting of YFP and a dihydrofolate reductase-thymidylate synthase selectable marker. The YFP is preceded by a ligation-independent cloning (LIC) cassette, which allows the insertion of PCR products containing complementary LIC sequences. We demonstrated that the {Delta}ku80 strain is more effective and efficient in integrating the YFP-tagged constructs into the correct locus than wild-type strain RH. We then selected several hypothetical proteins that were identified by a proteomic screen of excreted-secreted antigens and that displayed microarray expression profiles similar to known micronemal proteins, with the thought that these could potentially be new proteins with roles in cell invasion. We localized these hypothetical proteins by YFP fluorescence and showed expression by immunoblotting. Our findings demonstrate that the combination of the {Delta}ku80 strain and the pYFP.LIC constructs reduces both the time and cost required to determine localization of a new gene of interest. This should allow the opportunity for performing larger-scale studies of novel T. gondii genes.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, University of Michigan School of Medicine, 1150 W. Medical Center Dr., Ann Arbor, MI 48109. Phone: (734) 763-1928. Fax: (734) 764-3562. E-mail: vcarruth{at}umich.edu

{triangledown} Published ahead of print on 13 February 2009.

{dagger} Supplemental material for this article may be found at http://ec.asm.org/.

Eukaryotic Cell, April 2009, p. 530-539, Vol. 8, No. 4
1535-9778/09/$08.00+0     doi:10.1128/EC.00358-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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