Name: Genetic Manipulation in and Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii (Video) | JoVE Journal
Uploaded: 2013-07-12T13:00:00.000+00:00
Duration: 9 min 52 s
Description: Learn efficient gene replacement in Toxoplasma gondii using Δku80 strains with homologous recombination. This protocol enables precise genetic manipulation for functional genomic studies and vaccine development.

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

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<li>Donald, R. G., Roos, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+knock-outs+and+allelic+replacements+in+Toxoplasma+gondii%3A+HXGPRT+as+a+selectable+marker+for+hit-and-run+mutagenesis%5BTitle%5D)+AND+%22Molecular+and+Biochemical+Parasitology%22%5BJournal%5D)">Gene knock-outs and allelic replacements in Toxoplasma gondii: HXGPRT as a selectable marker for hit-and-run mutagenesis.</a> Molecular and Biochemical Parasitology. 91, 295-305 (1998).</li>
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<li>Oldenburg, K. R., Vo, K. T., Michaelis, S., Paddon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recombination-mediated+PCR-directed+plasmid+construction+in+vivo+in+yeast%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Recombination-mediated PCR-directed plasmid construction in vivo in yeast.</a> Nucleic Acids Res. 25, 451-452 (1997).</li>
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<li>Roos, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+genetic+tools+for+the+identification+and+analysis+of+drug+targets+in+Toxoplasma+gondii%5BTitle%5D)+AND+%22Curr.+Top.+Microbiol.+Immunol%22%5BJournal%5D)">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/pubmed?term=((Molecular+tools+for+genetic+dissection+of+the+protozoan+parasite+Toxoplasma+gondii%5BTitle%5D)+AND+%22Methods+in+Cell+Biology%22%5BJournal%5D)">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/pubmed?term=((Genetic+analysis+of+tachyzoite+to+bradyzoite+differentiation+mutants+in+Toxoplasma+gondii+reveals+a+hierarchy+of+gene+induction%5BTitle%5D)+AND+%22Molecular+Microbiology%22%5BJournal%5D)">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>
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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><tbody><tr><td>Overlap Primers</td><td>Integrated DNA Technologies</td><td></td><td>4 nmole Ultramer DNA oligos</td></tr><tr><td>Validation Primers</td><td>Integrated DNA Technologies</td><td></td><td>100 nmole DNA Oligo</td></tr><tr><td>Yeast Strain #90845</td><td>ATCC</td><td></td><td>Designation FY834</td></tr><tr><td>Shuttle Vector pRS416</td><td>ATCC</td><td>87521</td><td></td></tr><tr><td>DH10B &lt;em&gt;E. coli&lt;/em&gt;</td><td>Invitrogen</td><td>12033-015</td><td>SOC broth in kit with &lt;em&gt;E. coli&lt;/em&gt;</td></tr><tr><td>Resuspension Buffer</td><td>Qiagen</td><td></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></td><td></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></td><td></td></tr><tr><td>Maxiprep Kit</td><td>Qiagen</td><td>12662</td><td>QIAprep Spin Maxiprep Kit</td></tr><tr><td>25 cm&lt;sup&gt;2&lt;/sup&gt; Canted neck plug seal flask</td><td>Corning</td><td>430168</td><td></td></tr><tr><td>150 cm&lt;sup&gt;2&lt;/sup&gt; Canted Neck plug seal flask</td><td>Corning</td><td>430823</td><td></td></tr><tr><td>Human Foreskin Fibroblasts (HFF) cells</td><td>ATCC</td><td>SCRC1041</td><td></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></td><td></td></tr><tr><td>Xanthine (X)</td><td>Sigma</td><td></td><td></td></tr><tr><td>96-well Tissue Culture Tray</td><td>Corning Costar</td><td></td><td></td></tr><tr><td>24-well Tissue Culture Tray</td><td>Corning Costar</td><td></td><td></td></tr><tr><td>MEM Eagle Media</td><td>Lonza Biowhittaker</td><td>12-611F</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>26140-111</td><td></td></tr><tr><td>Antibiotic-Antimycotic (Anti-Anti)</td><td>Gibco</td><td>15240-062</td><td></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></td><td></td></tr><tr><td>6-thioxanthine</td><td>Acros Organics</td><td></td><td></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></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

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 pΔGOI targeting plasmid, and we note that continued selection often forces these episomes to integrate. We also observe some rare background in the type I Δku80 strain due to unintended integration of the pΔGOI 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 pΔGOI targeting plasmid may provide an alternative site for recombination in Δku80 strains2 that could give back an undesired background. In the type II Pru Δku80 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 Δku80 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 Δku80Δgoi::HXGPRT to delete HXGPRT from the strain is shown (Figure 3A). The primer pair used to validate the removal of HXGPRT from Δku80Δgra2::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 Δku80 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="Gene editing diagram with pRS416 plasmid, HxGPRT gene, primers, sequence table for PCR analysis." fo:content-width="6.5in" fo:src="/files/ftp_upload/50598/50598fig1highres.jpg" src="/files/ftp_upload/50598/50598fig1.jpg" data-altupdatedat="1787630408000" data-alt="Figure 1"> 
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 pΔROP1810. B. Overall strategy for designing a targeting DNA molecule. The backbone of pΔGOI 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="Gene editing diagram with PCR process, primers, and gel electrophoresis results for DNA analysis." fo:content-width="6.5in" fo:src="/files/ftp_upload/50598/50598fig2highres.jpg" src="/files/ftp_upload/50598/50598fig2.jpg" data-altupdatedat="1787630408000" data-alt="Figure 2"> 
Figure 2. Overview of the protocol for the deletion of a gene using HXGPRT. A. Disruption of a GOI in the Δku80Δhxgprt 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 pΔGOI. 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 pΔROP18 into Δku80Δhxgprt 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 Δku80Δhxgprt, 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="Genetic modification process diagram with PCR, flanking sequences, and gel electrophoresis results." fo:content-width="6.5in" fo:src="/files/ftp_upload/50598/50598fig3highres.jpg" src="/files/ftp_upload/50598/50598fig3.jpg" data-altupdatedat="1787630408000" data-alt="Figure 3"> 
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 Δku80Δgoi::HXGPRT using a ~1 Kbp 5' DNA target flank and a ~1 Kbp 3' DNA target flank on plasmid pΔGOIc. 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 Δku80Δgra2::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 pΔGOIc into Δku80Δgoi::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 Δku80Δgoi::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="Gene editing process diagram with GOI integration using HXGPRT and MPA selection strategy." fo:content-width="5in" fo:src="/files/ftp_upload/50598/50598fig4highres.jpg" src="/files/ftp_upload/50598/50598fig4.jpg" data-altupdatedat="1787630408000" data-alt="Figure 4"> 
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 pΔGOItag 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.

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.

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

The overall goal of this procedure is efficient and reliable gene replacement using type one and type two delta two 80 strains of Toxoplasmic ndii. This is accomplished by first constructing a targeting DNA molecule that fuses the HX GPRT selectable marker in the forward orientation between distinct five prime and three prime gene targeting DNA flanks. The second step of the procedure is to transfect the targeting DNA molecule into a type one or type two toxoplasma DYI strain.The third step is to patiently select for double crossover homologous recombination events and isolate individual clones. The final step is to validate the presence of the gene targeting event in the isolated mutant clones. Ultimately, results of this procedure can show the function of parasite genes by the analysis of targeted gene knockouts, gene replacements, and tag genes through a variety of downstream applications.The main advantage of this technique over existing methods using wild type strains of this parasite is that the type one and type two delta two 80 parasite backgrounds now enable extremely reliable selection of targeted double crossover homologous recombination events to produce precise genetically defined strains. This technique will greatly speed up the discovery of new targets for antitoxin plasmas drug therapy and the development of safe and effective attenuated vaccine strains, especially when paired with the well-conceived genomic resources located online@toxodb.org. Generally, individuals new to this method may struggle with engineering targeted DNA molecules or genetic manipulations with Toxoplasma Gandhi.However, patients practice and the design of reliable targeting molecules will ensure success. Begin the protocol by PCR amplifying the five prime and three prime genomic targeting flanks for the gene of interest from genomic DNA. Then PCR amplify the HX GPRT selectable marker with its five prime and three prime DHFR flanks from the HX GPRT CD NA mini gene cassette.Use yeast recombination or a similar method to fuse the 3D NA molecules in their correct five prime to three prime orientation with plasmid PRS four 16, thus generating plasmid p delta GOI that will target the deletion at the gene of interest. Next, prepare a 100 microliter sample containing 15 to 30 micrograms of linearized targeting plasmid for transfection into T gia 68 to 72 hours after inoculating a 25 centimeter flask of confluent HFF cells. Verify the presence of freshly lyed parasites by visually confirming that 90 to 95%of the HFF cells have been lyed viable parasites are essential for gene targeting.Agitate the viable parasites into solution by vigorously shaking the closed flask without splashing liquid onto the inside of the lid or the neck of the flask. Harvest the parasites from the flask and prepare them for transfection as described in the text protocol. Electroporated the targeting plasmid into the delta CO 80 toxoplasma strain.Transfer the electroporated mixture to a 150 centimeter flask containing confluent HFF cells and 30 milliliters of infection.Medium. Incubate the infected culture overnight at 37 degrees Celsius about 20 hours post transfection. Replace the medium with MPA plus X selection medium.Now allow the parasites to incubate at 37 degrees Celsius for about eight days.Undisturbed. Once plaques are visible, shake the flask to dislodge extracellular parasites from the monolayer generating a parasite solution. Transfer about half a milliliter of the parasite solution to a new 25 centimeter flask of confluent HFF cells containing five milliliters of MPA plus X selection medium.Designate this culture as PAs one A roughly three days later. Repeat the transfer of half a milliliter of the parasite solution from the original flask to another flask of HFF cells with the same selection media. Designate this culture as PAs one B over the next five days.Monitor passes one A and one B for pfu. Select either pass one A or one B for sub cloning based on an adequate number of greater than 25 zones of infection. Whenever parasites lyce out the HFF cells, pass the culture to a new 25 centimeter flask of confluent HFF cells with selection medium three weeks post transfection sub clone freshly laced parasites by diluting parasites to the appropriate concentration in MPA plus X medium, and evenly distributing the parasite solution into a 96 well tray containing confluent HFF cells.Prepare one tray at a concentration of one parasite per well and prepare a second tray at a concentration of two parasites per well. Incubate both trays at 37 degrees Celsius after the original transfection date. Do not subclone the parasites before day 20 for type one parasites or day 25 for type two parasites.Continue passing the 25 centimeter flask on a weekly schedule as described in the online text until the knockout strain has been validated. After about a week score the 96 well trays under 40 to 60 x magnification. Selecting only wells that contain a single PFU identified as a single zone of infection using a pipette set at 50 microliters.Mix the contents of these wells by directing fluid flow over the PFU to disperse the parasites around the well. Four to five days after mixing the selected wells, choose a dozen clones with lyed cells. Transfer 12 clones from the 96 well tray to 12 wells of a 24 Well tray Do this by scratching with a pipette tip across the bottom of the well and drawing up six microliters.Transfer the solution into a well of a 24 well tray with confluent HFF cells in one milliliter of MPA plus X selection. Medium incubate the 24 well tray at 37 degrees Celsius and monitor for lysis of the HFF cells. Passage the parasites from a lysed well in the 24 well tray on a weekly schedule.Use the scratch and draw technique to transfer two microliters of the parasite solution to a new plate with MPA and X selection. After passage, harvest the parasites from the original 24. Well tray and transfer to small tubes for DNA collection.To finish, use PCR to test for the deletion of the gene of interest to check the integration of the five prime and three prime target flanks. And to check for the HX GPRT selectable marker. This protocol is for the removal of the HX GPRT marker from the manipulated delta Q 80 strain and is outlined in the online text.P delta GOYC targets the removal of HX GPRT from the parasite by negative selection using six TX media. Then PCR is used to confirm the loss of HX GPRT as shown by a 1.2 Kilobase pair band where HX GPRT was not removed from the locus. There is a 3.4 kilobase pair band.The final part to this protocol, C terminal tagging of proteins is also described in detail in the online text after following the outlined protocol to make a targeted gene deletion, PCR was used to validate the deletion of a knockout of type one R 18. A low side that is generally difficult to target. PCR one shows the absence of the R 18 gene PCR product.PCR two shows the presence of the three prime R 18 genomic targeting flank PCR three and PCR four show the presence of the selectable marker properly integrated between the five prime and three prime genomic targeting flanks, which defines the R 18 deletion. In order to reuse the selectable marker to make subsequent deletions in a strain, the original HX GPRT must first be removed from the locus. After following the outlined protocol.HX GPRT was removed from the deleted GRA two locus in strain delta two 80 delta GRA two HX.G-P-R-T-P-C-R was used to confirm the loss of HX GPRT shown by a 1.2 KILOBASE per band. When H-X-G-P-R-T was not removed, there was a 3.4 kilobase per band Following the initial gene targeting step. Additional gene targeting methods can be applied to construct multiple knockouts in the same type one or type two strain, or to tag or complement a mutant strain to answer additional questions like, where does this protein localize in the cell?Or what is the key domain or activity of this protein or pathway that explains its biological function? This technique paves the way for researchers in the field to ramp up the exploration of parasite gene function in the model AP complex and parasite toxoplasma. Gandhi eye.Don't forget that working with toxoplasma can be hazardous and precautions such as following all BSL two guidelines should be maintained throughout the procedure.

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JoVE Journal

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