title: New selectable markers and single crossover integration for the highly versatile Plasmodium knowlesi transfection system.
abstract: Plasmodium knowlesi provides a highly versatile transfection system for malaria, since it enables rapid genetic modification of the parasite both in vivo as well as in vitro. However, it is not possible to perform multiple genetic manipulations within one parasite line because of a lack of selectable markers. In an effort to develop additional selectable markers for this parasite, positive and negative selectable markers that have recently been successfully used in Plasmodium falciparum were tested. It was shown that the positive selectable markers human dihydrofolate reductase (hdhfr), blasticidin S deaminase (bsd) and neomycin phosphotransferase II (neo) all conferred drug resistance to P. knowlesi when introduced as episomes. The plasmid containing the hdhfr selectable marker was not only successfully introduced as circular form, but also as linear fragment, demonstrating for the first time single crossover integration in P. knowlesi. Thymidine kinase was tested for its potential as negative selectable marker and it was shown that recombinant P. knowlesi parasites expressing thymidine kinase from episomes were highly sensitive to ganciclovir compared to wild-type P. knowlesi. The availability of new positive selectable markers and a strong candidate for a negative selectable marker for P. knowlesi, in combination with the opportunity to perform targeted single crossover integration in P. knowlesi, significantly increases the flexibility of this transfection system, making it one of the most versatile systems available for Plasmodium.
authors.affiliation: Department of Parasitology, Biomedical Primate Research Centre, P.O. Box 3306, 2280 GH, Rijswijk, The Netherlands.
authors.forename: Annemarie v d
authors.initials: Av
authors.lastname: Wel
authors.forename: Clemens H M
authors.initials: CH
authors.lastname: Kocken
authors.forename: Tamarah C
authors.initials: TC
authors.lastname: Pronk
authors.forename: Blandine
authors.initials: B
authors.lastname: Franke-Fayard
authors.forename: Alan W
authors.initials: AW
authors.lastname: Thomas
mesh_terms: D000619:Aminohydrolases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D004351:Drug Resistance / Q000235:genetics; D015774:Ganciclovir / Q000494:pharmacology; D015870:Gene Expression; D005819:Genetic Markers; D005839:Gentamicins / Q000494:pharmacology; D019868:Kanamycin Kinase / Q000235:genetics / Q000378:metabolism; D009705:Nucleosides / Q000494:pharmacology; D010957:Plasmids; D016790:Plasmodium knowlesi / Q000187:drug effects* / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D013937:Thymidine Kinase / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection / Q000379:methods*; D014227:Triazines / Q000494:pharmacology
chemical_list: D000981:Antiprotozoal Agents; D005819:Genetic Markers; D005839:Gentamicins; D009705:Nucleosides; D011994:Recombinant Proteins; D014227:Triazines; C006201:BRL 6231; C004500:blasticidin S; C010680:antibiotic G 418; D013762:Tetrahydrofolate Dehydrogenase; D013937:Thymidine Kinase; D019868:Kanamycin Kinase; D000619:Aminohydrolases; C019795:blasticidin S deaminase; D015774:Ganciclovir