Toxoplasma gondii is an important opportunistic infection of AIDS patients. Improved strategies and approaches are urgently needed to more effectively prevent and treat recurrent infections during AIDS. Due to the ease of in vitro culture and cloning, efficiency of transformation, abundance of established genetic tools, and availability of murine models of infection, Toxoplasma gondii is increasingly recognized as a model apicomplexan parasite. T. gondii is also being adopted as a surrogate parasite model to investigate biology of Cryptosporidium parvum, another very important opportunistic pathogen in the context of the AIDS pandemic, and other important apicomplexan parasites such as Plasmodium and Eimeria. To more effectively utilize T. gondii as a surrogate model organism for other parasites as well as to accelerate the development of improved prevention and treatment strategies for Toxoplasmosis, we propose to develop genetically improved model organism T gondii parasites with an enhanced efficiency of homologous recombination. Such model T. gondii parasites could be used to improve model systems for heterologous expression of genes from C. parvum or other pathogens and to develop genetically defined vaccine strains. T. gondii strains with enhanced homologous recombination can potentially accelerate functional genomics to more effectively identify and validate new drug targets, as well as to enhance aspects of biological discovery in the post-genomic era of T. gondii. To construct these novel T. gondii parasites, we propose to develop genetically defined parasite strains that are deficient in nonhomologous end-joining recombination repair based on the genetic construction of defined knockouts in the T. gondii gene homologs corresponding to KU70 and KU80. We predict that KU70 as well as KU80 knockouts in T. gondii will result in a phenotype of significantly enhanced homologous recombination. These studies will develop and validate strains that are likely to be useful model organisms for the more efficient construction of defined gene knockouts and gene manipulations in Toxoplasma gondii. ? ? ?
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