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The rodent parasite  Plasmodium chabaudi  is an important  in vivo  model of malaria. The ability to produce chronic infections makes it particularly useful for investigating the development of anti-  Plasmodium  immunity, as well as features associated with parasite virulence during both the acute and chronic phases of infection.  P. chabaudi  also undergoes asexual maturation (schizogony) and erythrocyte invasion in culture, so offers an experimentally-amenable  in vivo  to   in vitro  model for studying gene function and drug activity during parasite replication. To extend the usefulness of this model, we have further optimised transfection protocols and plasmids for  P. chabaudi  and generated stable, fluorescent lines that are free from drug-selectable marker genes. These mother-lines show the same infection dynamics as wild-type parasites throughout the lifecycle in mice and mosquitoes; furthermore, their virulence can be increased by serial blood passage and reset by mosquito transmission. We have also adapted the large-insert, linear  Plasmo GEM vectors that have revolutionised the scale of experimental genetics in another rodent malaria parasite and used these to generate barcoded  P. chabaudi  gene-deletion and –tagging vectors for transfection in our fluorescent  P. chabaudi  mother-lines. This produces a tool-kit of  P. chabaudi  lines, vectors and transfection approaches that will be of broad utility to the research community.

An enhanced toolkit for the generation of knockout and marker-free fluorescent Plasmodium chabaudi