Hostname: page-component-8448b6f56d-c47g7 Total loading time: 0 Render date: 2024-04-20T01:10:47.163Z Has data issue: false hasContentIssue false
  • English
  • Français

Advantages and disadvantages of conditional systems for characterization of essential genes in Toxoplasma gondii

Published online by Cambridge University Press:  13 June 2014

ELENA JIMÉNEZ-RUIZ Open the ORCID record for ELENA JIMÉNEZ-RUIZ [Opens in a new window] ,
ELEANOR H. WONG ,
GURMAN S. PALL  and
MARKUS MEISSNER
ELENA JIMÉNEZ-RUIZ
Affiliation:
Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
ELEANOR H. WONG
Affiliation:
Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
GURMAN S. PALL
Affiliation:
Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
MARKUS MEISSNER*
Affiliation:
Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK
*
*Corresponding author: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK. E-mail: markus.meissner@glasgow.ac.uk
Get access Rights & Permissions [Opens in a new window]

Summary

The dissection of apicomplexan biology has been highly influenced by the genetic tools available for manipulation of parasite DNA. Here, we describe different techniques available for the generation of conditional mutants. Comparison of the advantages and disadvantages of the three most commonly used regulation systems: the tetracycline inducible system, the regulation of protein stability and site-specific recombination are discussed. Using some previously described examples we explore some of the pitfalls involved in gene-function analysis using these systems that can lead to wrong or over-interpretation of phenotypes. We will also mention different options to standardize the application of these techniques for the characterization of gene function in high-throughput.

Keywords

apicomplexan parasitesgold-standard genetic toolsconditional mutantstetracycline inducible systemprotein destabilizationDiCre
Type
Special Issue Article
Information
Parasitology , Volume 141 , Special Issue 11: ApiCOWplexa 2013 – 2nd International Meeting on Apicomplexan Parasites in Farm Animals , September 2014 , pp. 1390 - 1398
Check for updates
Copyright
Copyright © Cambridge University Press 2014 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Agop-Nersesian, C., Pfahler, J., Lanzer, M. and Meissner, M. (2008). Functional expression of ribozymes in Apicomplexa: towards exogenous control of gene expression by inducible RNA-cleavage. International Journal for Parasitology 38, 673–681. doi: 10.1016/j.ijpara.2007.10.015.Google Scholar
Agop-Nersesian, C., Naissant, B., Ben Rached, F., Rauch, M., Kretzschmar, A., Thiberge, S., Menard, R., Ferguson, D. J., Meissner, M. and Langsley, G. (2009). Rab11A-controlled assembly of the inner membrane complex is required for completion of apicomplexan cytokinesis. PLOS Pathogen 5, e1000270. doi: 10.1371/journal.ppat.1000270.CrossRefGoogle ScholarPubMed
Agop-Nersesian, C., Egarter, S., Langsley, G., Foth, B. J., Ferguson, D. J. and Meissner, M. (2010). Biogenesis of the inner membrane complex is dependent on vesicular transport by the alveolate specific GTPase Rab11B. PLOS Pathogen 6, e1001029. doi: 10.1371/journal.ppat.1001029.CrossRefGoogle ScholarPubMed
Andenmatten, N., Egarter, S., Jackson, A. J., Jullien, N., Herman, J. P. and Meissner, M. (2013). Conditional genome engineering in Toxoplasma gondii uncovers alternative invasion mechanisms. Nature Methods 10, 125–127. doi: 10.1038/nmeth.2301.Google Scholar
Arakawa, H., Lodygin, D. and Buerstedde, J. M. (2001). Mutant loxP vectors for selectable marker recycle and conditional knock-outs. BMC Biotechnology 1, 7.Google Scholar
Armstrong, C. M. and Goldberg, D. E. (2007). An FKBP destabilization domain modulates protein levels in Plasmodium falciparum . Nature Methods 4, 1007–1009. doi: 10.1038/nmeth1132.Google Scholar
Aurrecoechea, C., Brestelli, J., Brunk, B. P., Fischer, S., Gajria, B., Gao, X., Gingle, A., Grant, G., Harb, O. S., Heiges, M., Innamorato, F., Iodice, J., Kissinger, J. C., Kraemer, E. T., Li, W., Miller, J. A., Nayak, V., Pennington, C., Pinney, D. F., Roos, D. S., Ross, C., Srinivasamoorthy, G., Stoeckert, C. J., Thibodeau, R., Treatman, C. and Wang, H. M. (2010). EuPathDB: a portal to eukaryotic pathogen databases. Nucleic Acids Research 38, D415–D419. doi: 10.1093/nar/gkp941.Google Scholar
Banaszynski, L. A., Chen, L. C., Maynard-Smith, L. A., Ooi, A. G. and Wandless, T. J. (2006). A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell 126, 995–1004. doi: 10.1016/j.cell.2006.07.025.Google Scholar
Bargieri, D. Y., Andenmatten, N., Lagal, V., Thiberge, S., Whitelaw, J. A., Tardieux, I., Meissner, M. and Menard, R. (2013). Apical membrane antigen 1 mediates apicomplexan parasite attachment but is dispensable for host cell invasion. Nature Communications 4, 2552. doi: 10.1038/ncomms3552.CrossRefGoogle ScholarPubMed
Boch, J., Scholze, H., Schornack, S., Landgraf, A., Hahn, S., Kay, S., Lahaye, T., Nickstadt, A. and Bonas, U. (2009). Breaking the code of DNA binding specificity of TAL-Type III effectors. Science 326, 1509–1512. doi: 10.1126/science.1178811.Google Scholar
Bouabe, H. and Okkenhaug, K. (2013). A protocol for construction of gene targeting vectors and generation of homologous recombinant embryonic stem cells. Methods in Molecular Biology 1064, 337–354. doi: 10.1007/978-1-62703-601-6_24.CrossRefGoogle ScholarPubMed
Brecht, S., Erdhart, H., Soete, M. and Soldati, D. (1999). Genome engineering of Toxoplasma gondii using the site-specific recombinase Cre. Gene 234, 239–247.Google Scholar
Breinich, M. S., Ferguson, D. J., Foth, B. J., van Dooren, G. G., Lebrun, M., Quon, D. V., Striepen, B., Bradley, P. J., Frischknecht, F., Carruthers, V. B. and Meissner, M. (2009). A dynamin is required for the biogenesis of secretory organelles in Toxoplasma gondii . Current Biology 19, 277–286. doi: 10.1016/j.cub.2009.01.039.CrossRefGoogle ScholarPubMed
Brooks, C. F., Johnsen, H., van Dooren, G. G., Muthalagi, M., Lin, S. S., Bohne, W., Fischer, K. and Striepen, B. (2010). The Toxoplasma apicoplast phosphate translocator links cytosolic and apicoplast metabolism and is essential for parasite survival. Cell Host and Microbe 7, 62–73. doi: 10.1016/j.chom.2009.12.002.Google Scholar
Buguliskis, J. S., Brossier, F., Shuman, J. and Sibley, L. D. (2010). Rhomboid 4 (ROM4) affects the processing of surface adhesins and facilitates host cell invasion by Toxoplasma gondii . PLOS Pathogens 6, doi: 10.1371/journal.ppat.1000858.Google Scholar
Carruthers, V. B., Giddings, O. K. and Sibley, L. D. (1999). Secretion of micronemal proteins is associated with Toxoplasma invasion of host cells. Cell Microbiology 1, 225–235.Google Scholar
Carvalho, T. G., Thiberge, S., Sakamoto, H. and Menard, R. (2004). Conditional mutagenesis using site-specific recombination in Plasmodium berghei . Proceedings of the National Academy of Sciences USA 101, 14931–14936. doi: 10.1073/pnas.0404416101.Google Scholar
Chong, W.-C., Basir, R. and Fei, Y. M. (2013). Eradication of malaria through genetic engineering: the current situation. Asian Pacific Journal of Tropical Medicine 6, 85–94. doi: 10.1016/s1995-7645(13)60001-2.Google Scholar
Collins, C. R., Das, S., Wong, E. H., Andenmatten, N., Stallmach, R., Hackett, F., Herman, J. P., Muller, S., Meissner, M. and Blackman, M. J. (2013). Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle. Molecular Microbiology 88, 687–701. doi: 10.1111/mmi.12206.Google Scholar
Combe, A., Giovannini, D., Carvalho, T. G., Spath, S., Boisson, B., Loussert, C., Thiberge, S., Lacroix, C., Gueirard, P. and Menard, R. (2009). Clonal conditional mutagenesis in malaria parasites. Cell Host and Microbe 5, 386–396. doi: 10.1016/j.chom.2009.03.008.CrossRefGoogle ScholarPubMed
Daher, W., Plattner, F., Carlier, M. F. and Soldati-Favre, D. (2010). Concerted action of two formins in gliding motility and host cell invasion by Toxoplasma gondii . PLOS Pathogen 6, e1001132. doi: 10.1371/journal.ppat.1001132.CrossRefGoogle ScholarPubMed
de Azevedo, M. F., Gilson, P. R., Gabriel, H. B., Simoes, R. F., Angrisano, F., Baum, J., Crabb, B. S. and Wunderlich, G. (2012). Systematic analysis of FKBP inducible degradation domain tagging strategies for the human malaria parasite Plasmodium falciparum . PLOS ONE 7, e40981. doi: 10.1371/journal.pone.0040981.CrossRefGoogle ScholarPubMed
Dvorin, J. D., Bei, A. K., Coleman, B. I. and Duraisingh, M. T. (2010). Functional diversification between two related Plasmodium falciparum merozoite invasion ligands is determined by changes in the cytoplasmic domain. Molecular Microbiology 75, 990–1006. doi: 10.1111/j.1365-2958.2009.07040.x.Google Scholar
Egarter, S., Andenmatten, N., Jackson, A. J., Whitelaw, J. A., Pall, G., Black, J. A., Ferguson, D. J. P., Tardieux, I., Mogilner, A. and Meissner, M. (2014). The Toxoplasma Acto-MyoA motor complex is important but not essential for gliding motility and host cell invasion. PLOS ONE, 9, e91819. doi: 10.1071/journal.pone.0091819.Google Scholar
Fox, B. A., Ristuccia, J. G., Gigley, J. P. and Bzik, D. J. (2009). Efficient gene replacements in Toxoplasma gondii strains deficient for nonhomologous end joining. Eukaryotic Cell 8, 520–529. doi: 10.1128/ec.00357-08.Google Scholar
Frenal, K., Polonais, V., Marq, J. B., Stratmann, R., Limenitakis, J. and Soldati-Favre, D. (2010). Functional dissection of the apicomplexan glideosome molecular architecture. Cell Host and Microbe 8, 343–357. doi: 10.1016/j.chom.2010.09.002.CrossRefGoogle ScholarPubMed
Frenal, K., Tay, C. L., Mueller, C., Bushell, E. S., Jia, Y., Graindorge, A., Billker, O., Rayner, J. C. and Soldati-Favre, D. (2013). Global analysis of apicomplexan protein S-acyl transferases reveals an enzyme essential for invasion. Traffic 14, 895–911. doi: 10.1111/tra.12081.CrossRefGoogle ScholarPubMed
Garcia-Otin, A. L. and Guillou, F. (2006). Mammalian genome targeting using site-specific recombinases. Frontiers in Bioscience 11, 1108–1136.CrossRefGoogle ScholarPubMed
Gossen, M. and Bujard, H. (1992). Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proceedings of the National Academy of Sciences USA 89, 5547–5551.Google Scholar
Hamann, L., Buss, H. and Tannich, E. (1997). Tetracycline-controlled gene expression in Entamoeba histolytica . Molecular and Biochemical Parasitology 84, 83–91.Google Scholar
Herm-Gotz, A., Agop-Nersesian, C., Munter, S., Grimley, J. S., Wandless, T. J., Frischknecht, F. and Meissner, M. (2007). Rapid control of protein level in the apicomplexan Toxoplasma gondii . Nature Methods 4, 1003–1005. doi: 10.1038/nmeth1134.Google Scholar
Huynh, M. H. and Carruthers, V. B. (2009). Tagging of endogenous genes in a Toxoplasma gondii strain lacking Ku80. Eukaryot Cell 8, 530–539. doi: 10.1128/ec.00358-08.Google Scholar
Iwamoto, M., Bjorklund, T., Lundberg, C., Kirik, D. and Wandless, T. J. (2010). A general chemical method to regulate protein stability in the mammalian central nervous system. Chemistry and Biology 17, 981–988. doi: 10.1016/j.chembiol.2010.07.009.Google Scholar
Jakot, D., Meissner, M., Sheiner, L., Soldati-Fevre, D. and Striepen, B. (2013). Genetic manipulation of Toxoplasma gondii . In Toxoplasma gondii. The Model Apicomplexan: Perspectives and Methods, 2nd Edn (ed. Weiss, L. M. and Kim, K.), pp. 577–611. Elsevier, Amsterdam, the Netherlands.Google Scholar
Jebiwott, S., Govindaswamy, K., Mbugua, A. and Bhanot, P. (2013). Plasmodium berghei calcium dependent protein kinase 1 is not required for host cell invasion. PLOS ONE 8, e79171. doi: 10.1371/journal.pone.0079171.Google Scholar
Jullien, N., Sampieri, F., Enjalbert, A. and Herman, J. P. (2003). Regulation of Cre recombinase by ligand-induced complementation of inactive fragments. Nucleic Acids Research 31. doi: 10.1093/nar/gng131.Google Scholar
Jullien, N., Goddard, I., Selmi-Ruby, S., Fina, J. L., Cremer, H. and Herman, J. P. (2007). Conditional transgenesis using dimerizable Cre (DiCre). PLoS ONE 2. doi: 10.1371/journal.pone.0001355.CrossRefGoogle ScholarPubMed
Kay, S. and Bonas, U. (2009). How Xanthomonas type III effectors manipulate the host plant. Current Opinion in Microbiology 12, 37–43. doi: 10.1016/j.mib.2008.12.006.Google Scholar
Kim, Y. G., Cha, J. and Chandrasegaran, S. (1996). Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proceedings of the National Academy of Sciences USA 93, 1156–1160. doi: 10.1073/pnas.93.3.1156.Google Scholar
Kolev, N. G., Tschudi, C. and Ullu, E. (2011). RNA interference in protozoan parasites: achievements and challenges. Eukaryotic Cell 10, 1156–1163. doi: 10.1128/ec.05114-11.CrossRefGoogle ScholarPubMed
Kremer, K., Kamin, D., Rittweger, E., Wilkes, J., Flammer, H., Mahler, S., Heng, J., Tonkin, C. J., Langsley, G., Hell, S. W., Carruthers, V. B., Ferguson, D. J. and Meissner, M. (2013). An overexpression screen of Toxoplasma gondii Rab-GTPases reveals distinct transport routes to the micronemes. PLOS Pathogen 9, e1003213. doi: 10.1371/journal.ppat.1003213.Google Scholar
Lacroix, C., Giovannini, D., Combe, A., Bargieri, D. Y., Spath, S., Panchal, D., Tawk, L., Thiberge, S., Carvalho, T. G., Barale, J. C., Bhanot, P. and Menard, R. (2011). FLP/FRT-mediated conditional mutagenesis in pre-erythrocytic stages of Plasmodium berghei . Nature Protocols 6, 1412–1428. doi: 10.1038/nprot.2011.363.CrossRefGoogle ScholarPubMed
Limenitakis, J. and Soldati-Favre, D. (2011). Functional genetics in Apicomplexa: potentials and limits. FEBS Letters 585, 1579–1588. doi: 10.1016/j.febslet.2011.05.002.Google Scholar
Lovett, J. L., Howe, D. K. and Sibley, L. D. (2000). Molecular characterization of a thrombospondin-related anonymous protein homologue in Neospora caninum . Molecular and Biochemical Parasitology 107, 33–43.CrossRefGoogle ScholarPubMed
Mali, P., Esvelt, K. M. and Church, G. M. (2013). Cas9 as a versatile tool for engineering biology. Nature Methods 10, 957–963. doi: 10.1038/nmeth.2649.Google Scholar
Meissner, M., Brecht, S., Bujard, H. and Soldati, D. (2001). Modulation of myosin A expression by a newly established tetracycline repressor-based inducible system in Toxoplasma gondii . Nucleic Acids Research 29, E115.Google Scholar
Meissner, M., Schluter, D. and Soldati, D. (2002). Role of Toxoplasma gondii myosin A in powering parasite gliding and host cell invasion. Science 298, 837–840. doi: 10.1126/science.1074553.Google Scholar
Meissner, M., Krejany, E., Gilson, P. R., de Koning-Ward, T. F., Soldati, D. and Crabb, B. S. (2005). Tetracycline analogue-regulated transgene expression in Plasmodium falciparum blood stages using Toxoplasma gondii transactivators. Proceedings of the National Academy of Sciences USA 102, 2980–2985. doi: 10.1073/pnas.0500112102.Google Scholar
Meissner, M., Breinich, M. S., Gilson, P. R. and Crabb, B. S. (2007). Molecular genetic tools in Toxoplasma and Plasmodium: achievements and future needs. Current Opinion in Microbiology 10, 349–356. doi: 10.1016/j.mib.2007.07.006.Google Scholar
Metzger, D., Clifford, J., Chiba, H. and Chambon, P. (1995). Conditional site-specific recombination in mammalian cells using a ligand-dependent chimeric Cre recombinase. Proceedings of the National Academy of Sciences USA 92, 6991–6995.Google Scholar
Muralidharan, V., Oksman, A., Iwamoto, M., Wandless, T. J. and Goldberg, D. E. (2011). Asparagine repeat function in a Plasmodium falciparum protein assessed via a regulatable fluorescent affinity tag. Proceedings of the National Academy of Sciences USA 108, 4411–4416. doi: 10.1073/pnas.1018449108.Google Scholar
Nain, V., Sahi, S. and Verma, A. (2010). CPP-ZFN: a potential DNA-targeting anti-malarial drug. Malaria Journal 9, 258. doi: 10.1186/1475-2875-9-258.Google Scholar
Nichols, B. A., Chiappino, M. L. and O'Connor, G. R. (1983). Secretion from the rhoptries of Toxoplasma gondii during host-cell invasion. Journal of Ultrastructure Research 83, 85–98.Google Scholar
Oberstaller, J., Joseph, S. J. and Kissinger, J. C. (2013). Genome-wide upstream motif analysis of Cryptosporidium parvum genes clustered by expression profile. BMC Genomics 14. doi: 10.1186/1471-2164-14-516.Google Scholar
Pan, Y. Z., Xiao, L., Li, A. S. S., Zhang, X., Sirois, P., Zhang, J. and Li, K. (2013). Biological and biomedical applications of engineered nucleases. Molecular Biotechnology 55, 54–62. doi: 10.1007/s12033-012-9613-9.Google Scholar
Pfander, C., Anar, B., Schwach, F., Otto, T. D., Brochet, M., Volkmann, K., Quail, M. A., Pain, A., Rosen, B., Skarnes, W., Rayner, J. C. and Billker, O. (2011). A scalable pipeline for highly effective genetic modification of a malaria parasite. Nature Methods 8, 1078. doi: 10.1038/nmeth.1742.Google Scholar
Pieperhoff, M. S., Schmitt, M., Ferguson, D. J. and Meissner, M. (2013). The role of clathrin in post-Golgi trafficking in Toxoplasma gondii . PLOS ONE 8, e77620. doi: 10.1371/journal.pone.0077620.Google Scholar
Pino, P. (2013). From technology to biology: a malaria genetic toolbox for the functional dissection of essential genes. Molecular Microbiology 88, 650–654. doi: 10.1111/mmi.12232.Google Scholar
Pino, P., Sebastian, S., Kim, E. A., Bush, E., Brochet, M., Volkmann, K., Kozlowski, E., Llinas, M., Billker, O. and Soldati-Favre, D. (2012). A tetracycline-repressible transactivator system to study essential genes in malaria parasites. Cell Host and Microbe 12, 824–834. doi: 10.1016/j.chom.2012.10.016.Google Scholar
Plageman, T. F. Jr. and Lang, R. A. (2012). Generation of an Rx-tTA: TetOp-Cre knock-in mouse line for doxycycline regulated Cre activity in the Rx expression domain. PLoS ONE 7, e50426. doi: 10.1371/journal.pone.0050426.Google Scholar
Prommana, P., Uthaipibull, C., Wongsombat, C., Kamchonwongpaisan, S., Yuthavong, Y., Knuepfer, E., Holder, A. A. and Shaw, P. J. (2013). Inducible knockdown of Plasmodium gene expression using the glmS ribozyme. PLoS ONE 8, e73783. doi: 10.1371/journal.pone.0073783.Google Scholar
Ramakrishnan, G., Vines, R. R., Mann, B. J. and Petri, W. A. Jr. (1997). A tetracycline-inducible gene expression system in Entamoeba histolytica . Molecular and Biochemical Parasitology 84, 93–100.Google Scholar
Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A. and Zhang, F. (2013). Genome engineering using the CRISPR-Cas9 system. Nature Protocols 8, 2281–2308. doi: 10.1038/nprot.2013.143.Google Scholar
Reichel, M. P. and Ellis, J. T. (2009). Neospora caninum – how close are we to development of an efficacious vaccine that prevents abortion in cattle? International Journal for Parasitology 39, 1173–1187.Google Scholar
Russo, I., Oksman, A., Vaupel, B. and Goldberg, D. E. (2009). A calpain unique to alveolates is essential in Plasmodium falciparum and its knockdown reveals an involvement in pre-S-phase development. Proceedings of the National Academy of Sciences USA 106, 1554–1559. doi: 10.1073/pnas.0806926106.Google Scholar
Sampson, T. R. and Weiss, D. S. (2014). Exploiting CRISPR/Cas systems for biotechnology. Bioessays 36, 34–38. doi: 10.1002/bies.201300135.Google Scholar
Santos, T. A., Portes Jde, A., Damasceno-Sa, J. C., Caldas, L. A., Souza, W., Damatta, R. A. and Seabra, S. H. (2011). Phosphatidylserine exposure by Toxoplasma gondii is fundamental to balance the immune response granting survival of the parasite and of the host. PLOS ONE 6, e27867. doi: 10.1371/journal.pone.0027867.Google Scholar
Sheiner, L., Demerly, J. L., Poulsen, N., Beatty, W. L., Lucas, O., Behnke, M. S., White, M. W. and Striepen, B. (2011). A systematic screen to discover and analyze apicoplast proteins identifies a conserved and essential protein import factor. PLOS Pathogens 7. doi: 10.1371/journal.ppat.1002392.Google Scholar
van Dooren, G. G., Reiff, S. B., Tomova, C., Meissner, M., Humbel, B. M. and Striepen, B. (2009). A novel dynamin-related protein has been recruited for apicoplast fission in Toxoplasma gondii . Current Biology 19, 267–276. doi: 10.1016/j.cub.2008.12.048.Google Scholar
van Poppel, N. F., Welagen, J., Duisters, R. F., Vermeulen, A. N. and Schaap, D. (2006). Tight control of transcription in Toxoplasma gondii using an alternative tet repressor. International Journal for Parasitology 36, 443–452.Google Scholar
Wang, J., Sarov, M., Rientjes, J., Fu, J., Hollak, H., Kranz, H., Xie, W., Stewart, A. F. and Zhang, Y. (2006). An improved recombineering approach by adding RecA to lambda Red recombination. Molecular Biotechnology 32, 43–53.Google Scholar
Wirtz, E. and Clayton, C. (1995). Inducible gene expression in trypanosomes mediated by a prokaryotic repressor. Science 268, 1179–1183.Google Scholar
Wirtz, E., Leal, S., Ochatt, C. and Cross, G. A. (1999). A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei . Molecular and Biochemical Parasitology 99, 89–101.Google Scholar
Zhang, N. Z., Chen, J., Wang, M., Petersen, E. and Zhu, X. Q. (2013). Vaccines against Toxoplasma gondii: new developments and perspectives. Expert Review of Vaccines 12, 1287–1299.Google Scholar