Tagging of endogenous genes in a Toxoplasma gondii strain lacking Ku80

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.

FIG. 1.

Targeted deletion of Ku80. (A) Generation of the Ku80 KO construct. Ku80 5′ and 3′ genomic flanks were amplified with overlaps to the dhfr-HXGPRT-dhfr selectable marker; the dhfr-HXGPRT-dhfr marker was amplified with overlaps to Ku80 flanks. A fusion PCR Δku80 product consisting of Ku80 flanks and the selectable marker was amplified. (B) Primer sets were used to evaluate the parental strain (RHhxgprt⁻) and Δku80 for replacement of the Ku80 gene with the selectable marker (a and a′), the presence or absence of the Ku80 gene (b and b′), and the presence or absence of the HXGPRT selectable marker (c and c′).

FIG. 2.

Endogenous gene tagging. (A) A pYFP.LIC.DHFR vector was generated that bore an upstream LIC cassette for cloning of genes of interest fused in frame to YFP, which was followed by the DHFR-TS 3′ UTR and a DHFR-TS cassette for selection of resistant parasites. (B) Schematic illustration of the single-crossover mechanism of integration of the YFP fusion protein to the 3′ end of a gene. (C) Positive control genes ACP (apicoplast), pCNA (nucleus), MIC3 (micronemes), and ROP1 (rhoptries) were cloned into the YFP expression vector and transfected into parasites, and localization was visualized by immunofluorescence with a GFP antibody. The DAPI staining in the ACP panel is pseudocolored with cyan to bring out the staining in the apicoplast. MIC2 staining was used to identify the micronemes and apical end of the parasite, and ROP2 staining was used to identify the rhoptries.

FIG. 3.

Optimization and efficiency of gene tagging. (A) Agarose gel showing plasmids in the forms supercoiled (SC), linearized within the targeting sequence (LN), or nicked/open circle (OC). (B) Linearization of the vector is necessary for efficient integration. Only the LN construct integrated into the homologous loci, whereas the SC and OC constructs did not integrate during the 30-day experiment. (C) Linearization site and efficiency of integration. Two restriction enzyme digestion sites were used, cutting 350 or 750 into the 1-kb targeting sequence. Both linearized constructs were shown to be equally effective in integration into the endogenous ACP locus. (D) Δku80 favors homologous recombination. pCNA-YFP, ACP-YFP, and MIC3-YFP constructs with either HXGPRT or DHFR-TS selectable markers were transfected into RH or Δku80, and YFP-positive parasites were enumerated over time and are expressed as a percentage of the total population. Note that the y-axis scale of the MIC3-YFP graph is smaller than for the pCNA-YFP and ACP-YFP graphs.

FIG. 4.

Localization of novel gene products by endogenous gene tagging. (A) YFP-tagged genes by immunofluorescence. (i) Microneme protein PLP1. (ii) 72.m00001, poly(ADP-ribose) glycohydrolase. (iii and iii′) 49.m03355 hypothetical protein. Arrowheads point to the YFP signal in apical poles of developing daughter cells. Inset: ring-like localization pattern. (iv) 83.m00006, SPATR-like protein. (v) 49.m00054, dystroglycan domain protein. (vi) 20.m03858 hypothetical protein. Scale bars were generated within the Zeiss Axio program. (B) Immunoblot of YFP-tagged genes probed with anti-GFP. Positions of molecular mass markers (in kDa) are indicated.