Abstract
The malaria parasite Plasmodium berghei is one of the main rodent malaria models. A shortcoming of this model parasite is its low flexibility in genetic manipulation. As this parasite cannot be continuously propagated in cell cultures, in vivo drug selection procedures are necessary to isolate genetic mutants. Drugs harmful to rodents therefore cannot be used for drug selection, which restricts the range of genetic manipulation. In this study, we addressed this problem by establishing a novel in vitro culture drug selection method, which we used in combination with other established methods to successfully isolate genetically manipulated parasites. The target mutants were enriched to the desired level within two weeks. We show that our system can also be used for sequential genetic manipulation of parasites carrying the traditionally used selection markers, demonstrate the procedure’s versatility, and show its use in isolating specific genetically manipulated parasites. This novel in vitro selection method increases the number of available selection markers, allowing more extensive genetic manipulation in malaria parasite research.
Highlights
Malaria is a global life-threatening disease caused by protozoan parasites of the genus Plasmodium
As puromycin is severely toxic to rodents and cannot be used for P. berghei in vivo, we developed a pac-puromycin system based on a novel short-term in vitro culture method
To determine whether pac can confer puromycin resistance to P. berghei, parasites transfected with pXL/hdhfr-pac-egfp carrying pac under the control of hsp[70] promoters (HSP70-PAC) were obtained using in vivo pyrimethamine selection
Summary
Malaria is a global life-threatening disease caused by protozoan parasites of the genus Plasmodium. In vivo drug selection procedures using rodents are required for the isolation of genetically manipulated P. berghei, as the parasite cannot be continuously propagated through in vitro cultures. Since this rules out drugs that are harmful to mammalian hosts, the use of popular mammalian drug selection marker genes is largely precluded[3]. This leads to difficulties in running sequential genetic manipulation experiments, such as phenotype rescue experiments using gene knockout (KO) parasites or the generation of multiple KO parasites[6,7,8].
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