This protocol describes a method of genetic transformation for the rodent malaria parasite Plasmodium berghei with a high transfection efficiency of 10(-3)-10(-4). It provides methods for: (i) in vitro cultivation and purification of the schizont stage;(ii) transfection of DNA constructs containing drug-selectable markers into schizonts using the nonviral Nucleofector technology; and (iii) injection of transfected parasites into mice and subsequent selection of mutants by drug treatment in vivo. Drug selection is described for two (antimalarial) drugs, pyrimethamine and WR92210. The drug-selectable markers currently in use are the pyrimethamine-resistant dihydrofolate reductase (dhfr) gene of Plasmodium or Toxoplasma gondii and the DHFR gene of humans that confer resistance to pyrimethamine and WR92210, respectively. This protocol enables the generation of transformed parasites within 10-15 d. Genetic modification of P. berghei is widely used to investigate gene function in Plasmodium, and this protocol for high-efficiency transformation will enable the application of large-scale functional genomics approaches.

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Sequestration of malaria-parasite-infected erythrocytes in the microvasculature of organs is thought to be a significant cause of pathology. Cerebral malaria (CM) is a major complication of Plasmodium falciparum infections, and PfEMP1-mediated sequestration of infected red blood cells has been considered to be the major feature leading to CM-related pathology. We report a system for the real-time in vivo imaging of sequestration using transgenic luciferase-expressing parasites of the rodent malaria parasite Plasmodium berghei. These studies revealed that: (i) as expected, lung tissue is a major site, but, unexpectedly, adipose tissue contributes significantly to sequestration, and (ii) the class II scavenger-receptor CD36 to which PfEMP1 can bind is also the major receptor for P. berghei sequestration, indicating a role for alternative parasite ligands, because orthologues of PfEMP1 are absent from rodent malaria parasites, and, importantly, (iii) cerebral complications still develop in the absence of CD36-mediated sequestration, dissociating parasite sequestration from CM-associated pathology. Realtime in vivo imaging of parasitic processes may be used to evaluate the molecular basis of pathology and develop strategies to prevent pathology.imaging ͉ Plasmodium ͉ P. berghei ͉ luciferase ͉ real-time in vivo imaging I nfected red blood cells (irbc) of many species of malaria parasites adhere to the endothelial cells of the microvasculature of numerous deep tissues (1, 2). Termed sequestration, this characteristic may facilitate parasite multiplication, avoiding removal of the irbc by the spleen (3, 4). In some parasite-host combinations, the process of sequestration is associated with pathogenesis, for example, Plasmodium falciparum in humans (1, 2, 5) and Plasmodium berghei in certain mouse strains (6, 7). Cerebral malaria (CM) is a major complication of P. falciparum infections, and the sequestration of irbc has been considered to be the major feature leading to CM-related pathology. Sequestration may lead to vascular obstruction, local endothelial cell activation, and the release of proinflammatory cytokines, resulting in damage to adjacent tissues (2, 7, 8). In P. falciparum, the class II scavenger receptor CD36 is a major endothelial receptor. CD36 is involved in not only the adherence of irbc (1, 9, 10) through specific domains of the surface variant antigen PfEMP-1 but also in the modulation of innate and adaptive immune responses (11,12). To date, most investigations of the dynamics of irbc-receptor interactions rely on in vitro observations with cultured cells and immobilized receptors (2). Despite the increase in knowledge of the molecules involved in the binding of irbc to endothelial cells, the specific interactions that lead to pathology have yet to be established. Infection with P. berghei in laboratory rodents is a well established model for the investigation of associations among CM, proinflammatory cytokines, and endothelial receptors involved in the sequestration of irbc, leukocytes, and platelet...