This work was supported by grants from the NIH to DJB (AI041930, AI073142, AI075931, and AI091461).

<ol>
	<li>Fox, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Type+II+Toxoplasma+gondii+KU80+knockout+strains+enable+functional+analysis+of+genes+required+for+cyst+development+and+latent+infection.">Type II Toxoplasma gondii KU80 knockout strains enable functional analysis of genes required for cyst development and latent infection.</a> Eukaryot. Cell. 10, 1193-1206 (2011).</li><li>Fox, B. A., Ristuccia, J. G., Gigley, J. P., Bzik, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+replacements+in+Toxoplasma+gondii+strains+deficient+for+nonhomologous+end+joining.">Efficient gene replacements in Toxoplasma gondii strains deficient for nonhomologous end joining.</a> Eukaryotic Cell. 8, 520-529 (2009).</li><li>Fox, B. A., Bzik, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avirulent+uracil+auxotrophs+based+on+disruption+of+orotidine-5'-monophosphate+decarboxylase+elicit+protective+immunity+to+Toxoplasma+gondii.">Avirulent uracil auxotrophs based on disruption of orotidine-5'-monophosphate decarboxylase elicit protective immunity to Toxoplasma gondii.</a> Infection and Immunity. 78, 3744-3752 (2010).</li><li>Hortua Triana, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+molecular+characterization+of+the+pyrimidine+biosynthetic+enzyme+dihydroorotate+dehydrogenase+from+Toxoplasma+gondii.">Biochemical and molecular characterization of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase from Toxoplasma gondii.</a> Molecular and Biochemical Parasitology. 184, 71-81 (2012).</li><li>Kim, K., Weiss, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma+gondii:+the+model+apicomplexan.">Toxoplasma gondii: the model apicomplexan.</a> Int. J. Parasitol. 34, 423-432 (2004).</li><li>Gajria, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ToxoDB:+an+integrated+Toxoplasma+gondii+database+resource.">ToxoDB: an integrated Toxoplasma gondii database resource.</a> Nucleic Acids research. 36, 553-556 (2008).</li><li>Kim, K., Weiss, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma:+the+next+100+years.">Toxoplasma: the next 100 years.</a> Microbes and infection / Institut Pasteur. 10, 978-984 (2008).</li><li>Butcher, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma+gondii+rhoptry+kinase+ROP16+activates+STAT3+and+STAT6+resulting+in+cytokine+inhibition+and+arginase-1-dependent+growth+control.">Toxoplasma gondii rhoptry kinase ROP16 activates STAT3 and STAT6 resulting in cytokine inhibition and arginase-1-dependent growth control.</a> PLoS pathogens. 7, e1002236 (2011).</li><li>Daher, W., Klages, N., Carlier, M. F., Soldati-Favre, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+characterization+of+Toxoplasma+gondii+formin+3,+an+actin+nucleator+dispensable+for+tachyzoite+growth+and+motility.">Molecular characterization of Toxoplasma gondii formin 3, an actin nucleator dispensable for tachyzoite growth and motility.</a> Eukaryot. Cell. 11, 343-352 (2012).</li><li>Fentress, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+immunity-related+GTPases+by+a+Toxoplasma+gondii-secreted+kinase+promotes+macrophage+survival+and+virulence.">Phosphorylation of immunity-related GTPases by a Toxoplasma gondii-secreted kinase promotes macrophage survival and virulence.</a> Cell Host Microbe. 8, 484-495 (2010).</li><li>Musiyenko, A., Majumdar, T., Andrews, J., Adams, B., Barik, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRMT1+methylates+the+single+Argonaute+of+Toxoplasma+gondii+and+is+important+for+the+recruitment+of+Tudor+nuclease+for+target+RNA+cleavage+by+antisense+guide+RNA.">PRMT1 methylates the single Argonaute of Toxoplasma gondii and is important for the recruitment of Tudor nuclease for target RNA cleavage by antisense guide RNA.</a> Cellular microbiology. 14, 882-901 (2012).</li><li>Straub, K. W., Peng, E. D., Hajagos, B. E., Tyler, J. S., Bradley, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+moving+junction+protein+RON8+facilitates+firm+attachment+and+host+cell+invasion+in+Toxoplasma+gondii.">The moving junction protein RON8 facilitates firm attachment and host cell invasion in Toxoplasma gondii.</a> PLos Pathog. 7, e1002007 (2011).</li><li>Szatanek, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cactin+is+essential+for+G1+progression+in+Toxoplasma+gondii.">Cactin is essential for G1 progression in Toxoplasma gondii.</a> Molecular Microbiology. 84, 566-577 (2012).</li><li>Donald, R. G., Carter, D., Ullman, B., Roos, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertional+tagging,+cloning,+and+expression+of+the+Toxoplasma+gondii+hypoxanthine-xanthine-guanine+phosphoribosyltransferase+gene.+Use+as+a+selectable+marker+for+stable+transformation.">Insertional tagging, cloning, and expression of the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase gene. Use as a selectable marker for stable transformation.</a> J. Biol. Chem. 271, 14010-14019 (1996).</li><li>Donald, R. G., Roos, D. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frozen+competent+yeast+cells+that+can+be+transformed+with+high+efficiency+using+the+LiAc/SS+carrier+DNA/PEG+method.">Frozen competent yeast cells that can be transformed with high efficiency using the LiAc/SS carrier DNA/PEG method.</a> Nature Protocols. 2, 1-4 (2007).</li><li>Oldenburg, K. R., Vo, K. T., Michaelis, S., Paddon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombination-mediated+PCR-directed+plasmid+construction+in+vivo+in+yeast.">Recombination-mediated PCR-directed plasmid construction in vivo in yeast.</a> Nucleic Acids Res. 25, 451-452 (1997).</li><li>Fox, B. A., Gigley, J. P., Bzik, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma+gondii+lacks+the+enzymes+required+for+de+novo+arginine+biosynthesis+and+arginine+starvation+triggers+cyst+formation.">Toxoplasma gondii lacks the enzymes required for de novo arginine biosynthesis and arginine starvation triggers cyst formation.</a> Int. J. Parasitol. 34, 323-331 (2004).</li><li>Roos, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+genetic+tools+for+the+identification+and+analysis+of+drug+targets+in+Toxoplasma+gondii.">Molecular genetic tools for the identification and analysis of drug targets in Toxoplasma gondii.</a> Curr. Top. Microbiol. Immunol. 219, 247-259 (1996).</li><li>Roos, D. S., Donald, R. G., Morrissette, N. S., Moulton, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+tools+for+genetic+dissection+of+the+protozoan+parasite+Toxoplasma+gondii.">Molecular tools for genetic dissection of the protozoan parasite Toxoplasma gondii.</a> Methods in Cell Biology. 45, 27-63 (1994).</li><li>Singh, U., Brewer, J. L., Boothroyd, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+analysis+of+tachyzoite+to+bradyzoite+differentiation+mutants+in+Toxoplasma+gondii+reveals+a+hierarchy+of+gene+induction.">Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction.</a> Molecular Microbiology. 44, 721-733 (2002).</li><li>vanden Hoff, M. J., Moorman, A. F., Lamers, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+in+'intracellular'+buffer+increases+cell+survival.">Electroporation in 'intracellular' buffer increases cell survival.</a> Nucleic Acids Research. 20, 2902 (1992).</li><li>Pfefferkorn, E. R., Bzik, D. J., Honsinger, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxoplasma+gondii:+mechanism+of+the+parasitostatic+action+of+6-thioxanthine.">Toxoplasma gondii: mechanism of the parasitostatic action of 6-thioxanthine.</a> Exp. Parasitol. 99, 235-243 (2001).</li><li>Mital, J., Meissner, M., Soldati, D., Ward, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+expression+of+Toxoplasma+gondii+apical+membrane+antigen-1+(TgAMA1)+demonstrates+that+TgAMA1+plays+a+critical+role+in+host+cell+invasion.">Conditional expression of Toxoplasma gondii apical membrane antigen-1 (TgAMA1) demonstrates that TgAMA1 plays a critical role in host cell invasion.</a> Mol. Biol. Cell. 16, 4341-4349 (2005).</li></ol>

The authors declare that they have no competing financial interests.

Here we provide a protocol for efficient gene targeting in &Delta;ku80 parasite strains to enable the efficient recovery of genetically manipulated progeny that possess targeted gene deletions, gene replacements, and/or tagged genes. The sequential execution of these methods provides a reliable approach for the isolation of parasite mutants that contain single or multiple targeted genetic manipulations1-3. While the generation of a targeted deletion depends on multiple factors, the use of the &Delta;ku80 strain with the presented strategy and protocol greatly increases the efficiency and ease of making precisely defined mutant strains of T. gondii for functional genomic studies.
The genome of T. gondii during asexual stages is haploid. Thus a consideration to make when planning to delete a gene is that the gene must not be essential in vitro. Nonetheless, the efficiency of targeting gene knockouts in &Delta;ku80 strains is extremely high, and this enables the isolation of mutant strains with significantly impaired replication rates. If a targeted gene is essential, parasites often cease to replicate during selection. Another critical factor in targeted genetic manipulation is that perfect homology with at least 620 bp of the genomic targeting flank is required to obtain detectable targeted integrations2. Longer regions of homology produce higher efficiencies of targeting and using targeting DNA flanks of approximately 1,000 bp is a reliable and efficient approach1-4. For this reason it is important that genomic targeting flanks are generated from the same strain of T. gondii (RH, Pru) as the strain that is being genetically manipulated. Lack of sufficient homology can frequently result in the targeted integration of one genomic targeting flank, but not the other. Incomplete integration or failure to obtain a knockout may also be due to episomal persistence. Immediately after transfection, all of the targeting molecules are nonintegrated episomes, and sometimes targeting molecules can persist as episomes for many generations. Using target DNA flanks of ~1 Kbp and continuing to pass parasites under selection prior to re-subcloning frequently resolves problems associated with episomal persistence.
Once a knockout is validated and the HXGPRT marker is removed, the gene targeting strategy can be repeated on a second GOI to generate multiple gene deletions in a single parasite strain1-3. The generation of deletions, replacements, or tagged genes can also be accomplished by using different selectable markers (bleomycin, CAT, pyrimethamine resistant DHFR, etc.). Although the CAT selectable marker currently is present in the type II &Delta;ku80 Pru genome1, this marker can be removed using the HXGPRT selection protocol described here. In our hands, HXGPRT selection is the safest and the most efficient marker with the highest targeting efficiency where a single-copy targeted insertion is sufficient for robust selection in MPA + X medium. HXGPRT also provides the only currently useful approach for targeted deletion of a selectable marker (Figure 3) using 6-thioxanthine (6TX) selection2,3. Thus this protocol provides the only current approach for developing defined mutant strains with several targeted genetic manipulations.
This protocol provides a valuable and efficient method for targeted genetic manipulation in &Delta;ku80 strains of Toxoplasma gondii and is applicable to analysis of a single gene, a family of genes, or genome-wide functional genomic studies. Prior to the availability of &Delta;ku80 strains1,2, these approaches were not widely feasible due to the inefficiency of these methods. Consequently, this protocol is widely applicable for targeted genetic manipulation of Toxoplasma gondii and is accessible to all investigators interested in addressing a range of questions on parasite biology, host response, and functional genomics.

Toxoplasma gondii is a common obligate intracellular protozoan parasite that frequently and chronically infects a wide range of animals and humans5. It is estimated that more than 1 billion humans are currently and chronically infected by this pathogen. In addition to the importance of disease caused by T. gondii infection, the increasing availability of experimental tools, powerful genomics resources6, ease of in vitro growth and excellent mouse models have made T. gondii a leading model system for the broader study of intracellular eukaryotic pathogens and other significant apicomplexan parasites that cause devastating diseases such as malaria (Plasmodium sp.) and Cryptosporidiosis (Cryptosporidium)5,7. A significant limitation of Toxoplasma gondii as a model organism has been the inefficient recovery of progeny that carry targeted genetic manipulations. This problem in gene targeting is due to a low frequency of homologous recombination relative to the very high frequency of nonhomologous recombination in wild-type strains of T. gondii even when extensive DNA homology is provided in DNA target molecules used in genetic studies2.
We recently genetically blocked the major pathway of nonhomologous recombination in type I and type II strains of Toxoplasma gondii by deleting the gene encoding the KU80 protein1,2. The resulting type I and type II &Delta;ku80 strains exhibit normal growth rates, size and behavior both in vitro and in vivo during tachyzoite and bradyzoite stages in vitro and in vivo, however, these strains exhibit essentially a 100% frequency of homologous recombination and this phenotype increases the likelihood of rapidly isolating desired progeny of targeted genetic manipulations by several hundred to several thousand-fold1,2,4. Recent community-wide use of type I and type II &Delta;ku80 strains has significantly ramped up the pace, the variety, as well as the success rate of targeted genetic approaches in T. gondii1-4,8-13. Here we describe a comprehensive protocol for targeted gene deletion, gene replacement, and gene tagging in &Delta;ku80 strains of T. gondii. We demonstrate how to design and reliably target genetic manipulations to specific genes or genetic loci in &Delta;ku80 strains of Toxoplasma gondii.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Overlap Primers</td>
			<td>Integrated DNA Technologies</td>
			<td>&nbsp;</td>
			<td>4 nmole Ultramer DNA oligos</td>
		</tr>
		<tr>
			<td>Validation Primers</td>
			<td>Integrated DNA Technologies</td>
			<td>&nbsp;</td>
			<td>100 nmole DNA Oligo</td>
		</tr>
		<tr>
			<td>Yeast Strain #90845</td>
			<td>ATCC</td>
			<td>&nbsp;</td>
			<td>Designation FY834</td>
		</tr>
		<tr>
			<td>Shuttle Vector pRS416</td>
			<td>ATCC</td>
			<td>87521</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DH10B E. coli</td>
			<td>Invitrogen</td>
			<td>12033-015</td>
			<td>SOC broth in kit with E. coli</td>
		</tr>
		<tr>
			<td>Resuspension Buffer</td>
			<td>Qiagen</td>
			<td>&nbsp;</td>
			<td>Buffer P1 in QIAprep Spin Miniprep Kit</td>
		</tr>
		<tr>
			<td>Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td>QIAprep Spin Miniprep Kit</td>
		</tr>
		<tr>
			<td>Glass Beads</td>
			<td>Scientific Industries</td>
			<td>SI-BG05</td>
			<td>0.5 mm acid-washed</td>
		</tr>
		<tr>
			<td>Qiacube Automated Robotic Work Station</td>
			<td>Qiagen</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electroporation Cuvette</td>
			<td>USA Scientific</td>
			<td>9104-5050</td>
			<td>2 mm-gap</td>
		</tr>
		<tr>
			<td>BTX600 electroporator</td>
			<td>BTX</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Maxiprep Kit</td>
			<td>Qiagen</td>
			<td>12662</td>
			<td>QIAprep Spin Maxiprep Kit</td>
		</tr>
		<tr>
			<td>25 cm2 Canted neck plug seal flask</td>
			<td>Corning</td>
			<td>430168</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>150 cm2 Canted Neck plug seal flask</td>
			<td>Corning</td>
			<td>430823</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Human Foreskin Fibroblasts (HFF) cells</td>
			<td>ATCC</td>
			<td>SCRC1041</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Swin-lok Filter Holder</td>
			<td>Whatman</td>
			<td>420200</td>
			<td>25-mm-diameter</td>
		</tr>
		<tr>
			<td>Membrane</td>
			<td>Whatman</td>
			<td>110612</td>
			<td>Nucleopore Track-etched Membrane 3 mm pore size, 25-mm-diameter</td>
		</tr>
		<tr>
			<td>Mycophenolic acid (MPA)</td>
			<td>Sigma</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Xanthine (X)</td>
			<td>Sigma</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>96-well Tissue Culture Tray</td>
			<td>Corning Costar</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>24-well Tissue Culture Tray</td>
			<td>Corning Costar</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>MEM Eagle Media</td>
			<td>Lonza Biowhittaker</td>
			<td>12-611F</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>26140-111</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (Anti-Anti)</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spin Column</td>
			<td>Primm Labs</td>
			<td>PAE-100</td>
			<td>Easy Clean DNA Extraction Spin Kit</td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>New England Biolabs</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>6-thioxanthine</td>
			<td>Acros Organics</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tissue DNA Minikit</td>
			<td>Qiagen</td>
			<td>51104</td>
			<td>QIAamp DNA Blood Mini Kit</td>
		</tr>
		<tr>
			<td>Cell Culture Freeze/Recovery Media</td>
			<td>Gibco</td>
			<td>126-48-010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Hyclone</td>
			<td>SH30028.02</td>
			<td>minus calcium, minus magensium</td>
		</tr>
	</tbody>
</table>

genetic manipulation in &delta;ku80 strains for functional genomic analysis of toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

A detailed template is provided for constructing a targeting plasmid to delete a gene, including the placement of restriction enzyme sites and generation of the primers that facilitate genetic targeting and validation of gene targeting, as well as plasmid construction for the subsequent deletion of HXGPRT in a single-step process (Figures 1A-C, Figure 2A, Figure 3A). A general schematic is presented for making a targeted gene deletion (Figure 2A), the primer pairs used to validate the deletion of a knockout, for example, type I rop18 (Figure 2B), and representative results of the PCR validation are shown (Figure 2C). This representative result is shown to illustrate the range of results that can be obtained at genetic loci that are relatively difficult to target. A successfully targeted gene deletion will result in the absence of the gene of interest PCR product (PCR 1), the presence of the 3' genomic targeting flank (PCR2), and the presence of the HXGPRT selectable marker properly integrated between the 5' and 3' genomic targeting flanks that define the deletion (PCR3 and PCR4). Clones 3, 4, 5, 6, 8, 9, and 11 are validated targeted gene deletions (knockouts) where HXGPRT has replaced the GOI (Figure 2C).
A clone that does not contain a gene deletion can be represented by a variety of banding patterns. Typically, a clone without a gene deletion will mirror the parental strain (parental pattern) with bands observed for PCR1 and PCR2, but not for PCR3 or PCR4 as seen for clones 1 and 2 (Figure 2C). This "parental pattern" arises from a few potential mechanisms. Occasionally, MPA resistant selected parasites carry nonintegrated persisting episomes of the p&Delta;GOI targeting plasmid, and we note that continued selection often forces these episomes to integrate. We also observe some rare background in the type I &Delta;ku80 strain due to unintended integration of the p&Delta;GOI targeting plasmid, which contains a HXGPRT cDNA expressed via DHFR 5' and 3' promoter elements, into either the DHFR locus or into the partially deleted HXGPRT locus14. While this rare event is a targeted integration, it was not the intended integration and serves as a key point to remember in designing any gene replacement strategy. DNA homology greater than 120 bp carried on your p&Delta;GOI targeting plasmid may provide an alternative site for recombination in &Delta;ku80 strains2 that could give back an undesired background. In the type II Pru &Delta;ku80 strain this background is reduced or nonexistent compared to type I because the HXGPRT selectable marker is based on type I sequences that have nucleotide polymorphisms when compared with type II DNA which greatly reduces this rare background in experiments using the type II Pru &Delta;ku80 strain1. If a gene locus is essential (cannot be deleted), or has an extremely low gene targeting frequency at the locus, or if persisting [nonintegrated] episomes are not easily eliminated via growth and selection, this parental pattern will dominate the pattern observed in MPA resistant clones. Alternatively, the targeting DNA molecule is sometimes observed to integrate at only the 5' or the 3' genomic targeting flank and a band is observed for either PCR3 or PCR4 (but not both) along with PCR1 and PCR2 as seen for clone 10 (5' integration) and clone 7 (3' integration) (Figure 2C). These patterns suggest the infrequent occurrence of a single cross over integration of the targeting plasmid at the locus that integrates HXGPRT but this targeted integration does not delete the gene of interest. This pattern (lanes 7 &amp; 10 in Figure 2C) emphasizes the need to report PCR data to verify that targeted integration occurred instead of a single homologous cross over and a second nonhomologous integration of the targeting molecule. Rarely, we observe a genetic mixture represented by clone 12 (Figure 2C) that represents both a parental pattern and a targeted deletion pattern. This pattern most likely arises on occasion from two parasite genotypes that are present at the same location in a cloning well.
A general schematic for the removal of HXGPRT from &Delta;ku80&Delta;goi::HXGPRT to delete HXGPRT from the strain is shown (Figure 3A). The primer pair used to validate the removal of HXGPRT from &Delta;ku80&Delta;gra2::HXGPRT (Figure 3B) amplifies a unique ~1.2 Kbp band in PCR5 as seen in clones 4, 7, 9, 11 and 12 (Figure 3C). If HXGPRT is not removed from the locus, a ~3.4 Kbp band is observed (clones 1, 2, 3, 6, 8, 10, and the parental control). Several mechanisms act to make the removal of HXGPRT (6TX selection) more challenging and less efficient than the integration of HXGPRT (MPA + X selection). MPA selection is simply more efficient than 6TX selection14,16. In addition, higher levels of HXGPRT expression are needed for 6TX selection than for MPA selections16,24. Consequently, mutations that may reduce HXGPRT enzyme activity or mutations or epigenetic changes that reduce the expression level of HXGPRT at a gene's locus can potentially abolish the effectiveness of 6TX selection. Even with these challenges of 6TX selection, the success rate of 6TX selection in &Delta;ku80 strains is greater than 90% on the first attempt1-3.
A general scheme for direct C-terminal tagging of protein-coding genes is shown (Figure 4). This scheme uses direct integration via double cross over homologous recombination of HXGPRT 3' of the tagged gene and also employs validation strategies similar to those described above. When a gene is suspected to be an essential gene this can be verified using a second transfection with an independently isolated targeting DNA plasmid. Alternative methods, such as schemes for regulated gene expression, are available for further verifying if a gene is essential25.
<img alt="Figure 1" fo:content-width="6.5in" fo:src="/files/ftp_upload/50598/50598fig1highres.jpg" src="/files/ftp_upload/50598/50598fig1.jpg" /> 
Figure 1. Overview of the design of a targeting DNA plasmid. A. Schematic for designing overlap primers used to PCR amplify the 5' and 3' target flanks with 33 bp overlaps for the pRS416 shuttle vector and HXGPRT minigene cassette. Primers depicted in the schematic were used to generate the 5' and 3' target flanks of p&Delta;ROP1810. B. Overall strategy for designing a targeting DNA molecule. The backbone of p&Delta;GOI is the pRS416 shuttle vector containing uracil (URA) and ampicillin (AMP) selectable markers. Inserted into pRS416 is a ~1 Kbp 5' DNA target flank amplified from genomic DNA with overlaps for the HXGPRT minigene cassette and pRS416 using the F1 and R1 primers, a ~1 Kbp 3' DNA target flank is amplified from genomic DNA with overlaps for the HXGPRT minigene cassette and pRS416 using the F2 and R2 primers and the HXGPRT minigene cassette. The following unique restriction enzyme digest sites are added to the primers: RE.X (restriction enzyme X cut site) in the F1 primer to cut the plasmid at the 5' end of the 5' DNA target flank, RE.Y in the R2 primer to cut the plasmid at the 3' end of the 3' DNA target flank and RE.Z in the R1 and F2 primers to excise the HXGPRT minigene cassette. C. Primer sequences used to generate the targeting construct for the deletion of rop18. The bold regions correspond to T. gondii genomic sequence, and the nonbold regions correspond to the restriction enzyme sites and sequences that overlap with pRS416 and the HXGPRT minigene cassette. The HX_F and HX_R primer pair amplifies the HXGPRT minigene cassette14. Primers read from 5' to 3'. Table adapted from Fentress et al10. <a href="https://www.jove.com/files/ftp_upload/50598/50598fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" fo:content-width="6.5in" fo:src="/files/ftp_upload/50598/50598fig2highres.jpg" src="/files/ftp_upload/50598/50598fig2.jpg" /> 
Figure 2. Overview of the protocol for the deletion of a gene using HXGPRT. A. Disruption of a GOI in the &Delta;ku80&Delta;hxgprt strain by a double crossover homologous recombination event using a ~1 Kbp 5' DNA target flank and a ~1 Kbp 3' DNA target flank on plasmid p&Delta;GOI. A PCR strategy using PCR1, PCR2, PCR3, and PCR4 (not to scale) is shown that enables genotype verification to validate clones with targeted integration of HXGPRT and deletion of the GOI locus. B. Representative primer pairs designed to validate the deletion of rop18. ROP18_DF and ROP18_DR amplify PCR1, ROP18_ExF and ROP18_CxR amplify PCR2, ROP18_CxF and DHFR_CxR amplify PCR3 and DHFR_CxF and ROP18_CxR amplify PCR4 (see Figure 2A). Primers read from 5' to 3'. Table adapted from Fentress et al10. C. Representative results of validation of a targeted gene deletion (rop18) using HXGPRT. Following transfection of plasmid p&Delta;ROP18 into &Delta;ku80&Delta;hxgprt and selection in MPA+X, MPA+X resistant clones were assayed for the deletion of rop18. The parental strain control is positive for PCR 1(~400 bp) and PCR 2 (~650 bp) product and negative for PCR 3 (~1,200 bp) and PCR 4 (~1,300 bp) product. A targeted GOI knockout is positive for the PCR2, PCR3 and PCR4 products and is negative for the PCR 1 product (see Figure 2A). Shown are representative panels of the results of PCR1 and PCR2 (top panel), PCR3 (middle panel), and PCR4 (bottom panel). Clones 3, 4, 5, 6, 8, 9 and 11 exhibit the correct banding pattern of PCR 1, PCR2, PCR3 and PCR4 that defines a targeted gene deletion event in the clone. Clones 1, 2, 7, 10 and 12 are not gene deletions, and correspond to other potential representative results. (C = parental control strain &Delta;ku80&Delta;hxgprt, M = size markers). <a href="https://www.jove.com/files/ftp_upload/50598/50598fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 3" fo:content-width="6.5in" fo:src="/files/ftp_upload/50598/50598fig3highres.jpg" src="/files/ftp_upload/50598/50598fig3.jpg" /> 
Figure 3. Overview of the protocol for re-targeting the gene of interest to delete HXGPRT. A. Strategy for removal of the HXGPRT selectable marker by a double crossover homologous recombination event in the strain &Delta;ku80&Delta;goi::HXGPRT using a ~1 Kbp 5' DNA target flank and a ~1 Kbp 3' DNA target flank on plasmid p&Delta;GOIc. The PCR strategy for genotype verification is depicted using a primer pair to assay for a PCR product (PCR5) that spans the deletion (not to scale). B. A representative primer pair designed to validate the removal of the HXGPRT selectable marker from the gra2 locus in strain &Delta;ku80&Delta;gra2::HXGPRT. Primers GRA2 _CLF and GRA2_CxR amplify PCR5. Primers read from 5' to 3'. C. Representative panel of 6TX-resistant clones that may be obtained following transfection of plasmid p&Delta;GOIc into &Delta;ku80&Delta;goi::HXGPRT and selection in 6TX. Clones 4, 7, 9, 11 and 12 exhibit the correct banding pattern of PCR5 that corresponds to targeted deletion of the HXGPRT marker (~1.2 Kbp product that spans the 3' flank with slight overlap with the 5' flank and outside the 3' flank). Clones 1, 2, 3, 6, 8, and 10 (this band is light) represent the expected banding pattern of around ~3.4 Kbp that corresponds to the parental clone genotype, even though these clones exhibited a degree of resistance to 6TX that permitted their selection (this phenotype can occasionally arise spontaneously by shut-down of HXGPRT expression (see note in protocol 2.1 (step 8) and Representative Results section). (C = parental control strain &Delta;ku80&Delta;goi::HXGPRT, M = size markers). <a href="https://www.jove.com/files/ftp_upload/50598/50598fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/50598/50598fig4highres.jpg" src="/files/ftp_upload/50598/50598fig4.jpg" /> 
Figure 4. Design of a targeting DNA molecule to tag the C-terminal end of a protein. The strategy is based on the ability of the HXGPRT marker to also function as a 3' downstream regulatory region. The backbone of p&Delta;GOItag is the pRS416 shuttle vector containing uracil (URA) and ampicillin (AMP) selectable markers. Inserted into pRS416 is a ~1 Kbp 5' DNA target flank amplified from genomic DNA with overlaps for the HXGPRT minigene cassette and pRS416 using the Fgoi and Rtag primers, a ~1 Kbp 3' DNA target flank is amplified from genomic DNA with overlaps for the HXGPRT minigene cassette and pRS416 using the F2 and R2 primers and the HXGPRT minigene cassette. The 5' DNA target flank replaces the termination codon with the tag (HA, Myc, His, etc.) followed by a replacement termination codon. MPA + X selection integrates the C-terminal tag and a downstream HXGPRT, and moves the 3' UTR to a position following the HXGPRT marker. The following unique restriction enzyme digest sites are added to the primers: RE.X (restriction enzyme X cut site) in the Fgoi primer to cut the plasmid at the 5' end of the 5' DNA target flank, and RE.Y in the R2 primer to cut the plasmid at the 3' end of the 3' DNA target flank, and RE.Z in the Rtag and F2 primers to excise the HXGPRT minigene cassette for use for plasmid construction to re-target the tagged gene locus to delete the HXGPRT marker which will restore the placement of the 3' UTR.

Watch this Scientific Journal Video about Genetic Manipulation in ÃŽâ€ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii at JoVE.com

Genetic Manipulation in ÃƒÅ½Ã¢â‚¬Âku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

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