Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle.

Collins CR1, Das S, Wong EH, Andenmatten N, Stallmach R, Hackett F, Herman JP, Müller S, Meissner M, Blackman MJ.
Source: Mol Microbiol
Publication Date: (2013)
Issue: 88(4): 687-701
Research Area:
Basic Research
Cells used in publication:
Plasmodium falciparum
Species: unicellular
Tissue Origin:
4D-Nucleofectorâ„¢ X-Unit
For introduction of transfection constructs, mature schizonts were enriched from highly synchronous cultures using Percoll (GE Healthcare) as described previously (Harris et al., 2005), and transfected by electroporation with 10 mg of circular plasmid DNA using the Amaxa 4D electroporator (Lonza) and the P3 Primary cell 4D Nucleofector X Kit L (Lonza) and program FP158, exactly as recently described for P. knowlesi (Moon et al., 2013)
Asexual blood stages of the malaria parasite, which cause all the pathology associated with malaria, can readily be genetically modified by homologous recombination, enabling the functional study of parasite genes that are not essential in this part of the life cycle. However, no widely applicable method for conditional mutagenesis of essential asexual blood-stage malarial genes is available, hindering their functional analysis. We report the application of the DiCre conditional recombinase system to Plasmodium falciparum, the causative agent of the most dangerous form of malaria. We show that DiCre can be used to obtain rapid, highly regulated site-specific recombination in P. falciparum, capable of excising loxP-flanked sequences from a genomic locus with close to 100% efficiency within the time-span of a single erythrocytic growth cycle. DiCre-mediated deletion of the SERA5 3' UTR failed to reduce expression of the gene due to the existence of alternative cryptic polyadenylation sites within the modified locus. However, we successfully used the system to recycle the most widely used drug resistance marker for P. falciparum, human dihydrofolate reductase, in the process producing constitutively DiCre-expressing P. falciparum clones that have broad utility for the functional analysis of essential asexual blood-stage parasite genes.