Abstract
Background: Malaria research is greatly dependent on and has drastically advanced with the possibility of genetically modifying Plasmodium parasites. The commonly used transfection protocol by Janse and colleagues utilizes blood stage-derived Plasmodium berghei schizonts that have been purified from a blood culture by density gradient centrifugation. Naturally, this transfection protocol depends on the availability of suitably infected mice, constituting a time-based variable. In this study, the potential of transfecting liver stage-derived merozoites was explored. In cell culture, upon merozoite development, infected cells detach from the neighbouring cells and can be easily harvested from the cell culture supernatant. This protocol offers robust experimental timing and temporal flexibility. Methods: HeLa cells are infected with P. berghei sporozoites to obtain liver stage-derived merozoites, which are harvested from the cell culture supernatant and are transfected using the Amaxa Nucleofector® electroporation technology. Results: Using this protocol, wild type P. berghei ANKA strain and marker-free PbmCherryHsp70-expressing P. berghei parasites were successfully transfected with DNA constructs designed for integration via single- or double-crossover homologous recombination. Conclusion: An alternative protocol for Plasmodium transfection is hereby provided, which uses liver stage-derived P. berghei merozoites for transfection. This protocol has the potential to substantially reduce the number of mice used per transfection, as well as to increase the temporal flexibility and robustness of performing transfections, if mosquitoes are routinely present in the laboratory. Transfection of liver stage-derived P. berghei parasites should enable generation of transgenic parasites within 8-18 days.
Original language | English (US) |
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Article number | 305 |
Journal | Malaria journal |
Volume | 16 |
Issue number | 1 |
DOIs | |
State | Published - Aug 1 2017 |
Funding
We thank Chris J. Janse and Shahid M Khan for providing the selection‑marker‑ free mCherry and the mCherryLuc expressing parasites. We thank Tobias Spielmann for providing the PfSBP1 and PbSBP1KO constructs. Images were acquired on equipment supported by the Microscopy Imaging Centre of the University of Bern. Financial support came from the Swiss National Founda‑ tion (310030_140691/1) (VTH) and the EVIMalaR EU network (FP7/2007‑2013) (MDN and VTH).
Keywords
- Liver stage-derived merozoites
- Plasmodium berghei
- Transfection
ASJC Scopus subject areas
- Parasitology
- Infectious Diseases