Experimental genetic crosses for malaria research

PROJECT SUMMARY A concrete way to map genotypes that cause Plasmodium falciparum drug resistance phenotypes is by performing well thought out experimental genetic crosses between drug susceptible and resistant strains, isolating recombinant progeny and then using quantitative trait loci mapping to link genotype to phenotype. As an alternative, bulk segregant analysis under drug pressure can be used to pinpoint loci of interest. P. falciparum genetic crosses were initially carried out in splenectomized chimpanzees, but NIH has banned chimpanzee research for financial and ethical reasons. To overcome this, we have developed and improved on a human-liver chimeric mouse model (the FRG NOD huHep mouse) to replace the chimpanzee for the generation of recombinant progeny from P. falciparum genetic crosses. The FRG NOD mouse lacks the fumaryl acetoacetate hydrolase gene (F designation) and this causes hepatocyte cell death. However, hepatocyte death is controlled with the drug nitisinone. Since only mouse hepatocytes lack fumaryl acetoacetate hydrolase, this enables repopulation of the mouse with human hepatocytes over time with on-off drug use to control the death of mouse hepatocytes and their replacement with human hepatocytes. In close collaboration with the Yecuris Corporation, who creates the FRG NOD huHep mouse, we ensure that the mice we use for our studies have maximal human hepatocyte chimerism and are susceptible to P. falciparum sporozoite infection. Additionally, the mice are able to maintain a human red blood cell (huRBC) population after huRBC infusion and this allows for P. falciparum liver stage-to-blood stage transition in the mouse. Following blood removal, the in vitro expansion of asexual P. falciparum blood stages allows for bulk segregant analysis as well as downstream cloning and then –omics analyses and phenotypic analyses of recombinant progeny. We have clearly demonstrated our ability to use the FRG NOD huHep/huRBC mouse for the generation of recombinant progeny from experimental crosses and Core A will continue to produce recombinant progeny from a further ten well-conceived experimental genetic crosses between P. falciparum drug resistant and drug susceptible strains as part of this P01 renewal. The phenotyping of bulk and isolated progeny supplied by Core A and downstream mapping of genetic loci responsible for observed phenotypes are integral parts of RP01, RP02 & RP03 and, as such, Core A is the linchpin of this P01 renewal. Successful creation of progeny for this P01 will further our understanding of the spread of artemisinin drug resistance, which has now been documented to have occurred independently in Africa and is of grave concern to malaria control and elimination efforts.