Article

Tagging of Endogenous Genes in a Toxoplasma gondii Strain Lacking Ku80

Department of Microbiology and Immunology, University of Michigan School of Medicine, Ann Arbor, MI 48109, USA.
Eukaryotic Cell (Impact Factor: 3.18). 03/2009; 8(4):530-9. DOI: 10.1128/EC.00358-08
Source: PubMed

ABSTRACT

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 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 Deltaku80 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 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 allow the opportunity for performing larger-scale studies of novel T. gondii genes.

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Available from: Vern B Carruthers
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    • "type I RH tachyzoites and their transgenic derivatives were grown in human foreskin fibroblasts (HFFs) as described inRoos et al. (1994). The following lines have been described before: RH::Δku80 (Huynh and Carruthers, 2009) was kindly shared by Vern Carruthers (University of Michigan), TATi::Δku80 (Sheiner et al., 2011) was kindly shared by Boris Striepen (University of Georgia), and TgNuf2-cKD was generated by Farrell and Gubbels (2014). The TgEB1-KO line was established by replacing the endogenous locus with a 1.5-kb PCR product composed of an HXGPRT drug-selectable cassette and the TgEB1-cKD line with a 4.1-kb PCR product composed of DHFR-Tet7Sag4-TgEB1-(CDS)-2xMyc. "
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    • "All rights reserved. DHFR vector (Bougdour et al., 2013) using the LIC cloning method as described (Huynh et al., 2009). Approximately 20 µg of the resulting vector was linearized within the region of homology with AvrII before transfection. "
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    • "The parasite strain RH / ku80 - / hxgprt - ( Huynh and Carruthers , 2009 ) ( ATCC : PRA319 ) was used as the parental strain of the trans - genic parasites throughout this study . Parasites were maintained and serially passaged to new host Vero cells as described else - where ( Roos et al . "
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