Eukaryot Cell. 2009 Feb 13. [Epub ahead of print]
Tagging of endogenous genes in a Toxoplasma gondii strain lacking Ku80
Huynh MH, Carruthers VB.
Department of Microbiology and Immunology, University of Michigan School of Medicine, 1150 W. Medical Center Dr., Ann Arbor, MI 48109 USA.
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 Deltaku80 strain. Ku80 is involved in DNA strand repair and non-homologous 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 DHFR-TS selectable marker. The YFP is preceded by a LIC (ligation-independent cloning) cassette, which allows the insertion of PCR products containing complementary LIC sequences. We demonstrate that the Deltaku80 strain is more effective and efficient in integrating the YFP-tagged genes into the correct locus compared to the wild-type strain RH. We then selected several hypothetical proteins that were identified by a proteomic screen of excreted-secreted antigens (ESA) 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 show expression by immunoblotting. Our findings demonstrate that the combination of the Deltaku80 strain and the pYFP.LIC constructs reduces both the time and cost required to determine localization of a new gene of interest. This should open the opportunity for performing larger scale studies of novel T. gondii genes.
PMID: 19218426 [PubMed - as supplied by publisher]