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Selection by flow-sorting of genetically transformed, GFP-expressing blood stages of the rodent malaria parasite, Plasmodium berghei

Nature Protocols volume 1, pages 614–623 (2006)Cite this article

Abstract

This protocol describes a methodology for the genetic transformation of the rodent malaria parasite Plasmodium berghei and the subsequent selection of transformed parasites expressing green fluorescent protein (GFP) by flow-sorting. It provides methods for: transfection of the schizont stage with DNA constructs that contain gfp as the selectable marker; selection of fluorescent mutants by flow-sorting; and injection of flow-sorted, GFP-expressing parasites into mice and the subsequent collection of transformed parasites. The use of two different promoters for the expression of GFP is described; these two promoters require slightly different procedures for the selection of mutants. The protocol enables the collection of transformed parasites within 10–12 days after transfection. The genetic modification of P. berghei is widely used to investigate gene function in Plasmodium sp. The application of flow-sorting to the selection of transformed parasites increases the possibilities of parasite mutagenesis, by effectively expanding the range of selectable markers.

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Figure 1: The procedure for the selection of fluorescent, GFP-expressing blood stages of Plasmodium berghei by flow-sorting.
Figure 2: A 24-well culture plate containing infected tail blood in culture medium, placed in a glass desiccator (candle jar).
Figure 3: Analysis of GFP fluorescence intensity by flow cytometry of the blood stages of transgenic Plasmodium berghei lines.

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Acknowledgements

We would like to thank J. Ramesar, H. Kroeze and S. Khan for their critical comments, and M. van der Keur and R. van der Linden for their help with the development of the flow-sorting procedure.

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  1. Leiden, 2300, RC, The Netherlands, P.O. Box 9600

    Chris J Janse, Blandine Franke-Fayard & Andrew P Waters

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  1. Chris J Janse
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  2. Blandine Franke-Fayard
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  3. Andrew P Waters
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Correspondence to Chris J Janse.

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Janse, C., Franke-Fayard, B. & Waters, A. Selection by flow-sorting of genetically transformed, GFP-expressing blood stages of the rodent malaria parasite, Plasmodium berghei. Nat Protoc 1, 614–623 (2006). https://doi.org/10.1038/nprot.2006.88

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