Transgenesis work used the P. falciparum 3D7-derived DiCre-expressing clone 1G5DiCre (14 (link)), also known as 1G5DC. Asexual blood-stage parasites were continuously cultured in complete medium (cRPMI; Roswell Park Memorial Institute [RPMI] 1640 medium containing Albumax [Gibco, Grand Island, NY]) and synchronized with 5% d -sorbitol (27 (link)). Transfection of 1G5DC was as previously described (28 (link)). Briefly, mature schizonts were enriched using Percoll (29 (link)) (GE Healthcare, Chicago, IL) and electroporated using an Amaxa 4D electroporator and P3 Primary cell 4D Nucleofector X kit L (Lonza, Basel, Switzerland) (14 (link), 30 (link)). Drug selection with cRPMI supplemented with 2.5 nM WR99210 was initiated after 48 h posttransfection to allow selection of parasites transfected with plasmids carrying the human dihydrofolate reductase gene (dhfr) selectable marker. Integrant parasite clones for version 1 (v1) (3E10 and 7C4) and v2 (B11) were isolated by limiting dilution.
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Transgenesis of Plasmodium falciparum
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Corresponding Organization : National Institute of Allergy and Infectious Diseases
Other organizations : The Francis Crick Institute, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Ehime University, London School of Hygiene & Tropical Medicine
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Variable analysis
independent variables
- Use of DiCre-expressing clone 1G5DiCre (also known as 1G5DC) of the P. falciparum 3D7 strain
- Transfection of 1G5DC parasites
- Drug selection with 2.5 nM WR99210
- Integrant parasite clones for version 1 (v1) (3E10 and 7C4) and v2 (B11)
- Continuous culture of asexual blood-stage parasites in complete medium (cRPMI)
- Synchronization of parasites with 5% d-sorbitol
- Enrichment of mature schizonts using Percoll
- Electroporation using an Amaxa 4D electroporator and P3 Primary cell 4D Nucleofector X kit L
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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