10.17863/CAM.57160
Carrasquilla, Manuela
0000-0002-7953-4376
Adjalley, Sophie
Sanderson, Theo
Marin-Menendez, Alejandro
Coyle, Rachael
Montandon, Ruddy
Rayner, Julian C
0000-0002-9835-1014
Pance, Alena
0000-0002-9017-2644
Lee, Marcus CS
Defining multiplicity of vector uptake in transfected Plasmodium parasites.
Springer Science and Business Media LLC
2020
Biological Transport
Calmodulin
Clone Cells
DNA Barcoding, Taxonomic
DNA, Recombinant
Electroporation
Erythrocytes
Flow Cytometry
Gene Library
Genetic Vectors
Humans
Luminescent Proteins
Plasmids
Plasmodium falciparum
Plasmodium knowlesi
Promoter Regions, Genetic
Species Specificity
Transfection
Apollo - University of Cambridge Repository
University of Cambridge
013meh722
2020-09-09
2020-09-09
2020-07-02
eng
Article
https://www.repository.cam.ac.uk/handle/1810/310074
10.1038/s41598-020-67791-z
Attribution 4.0 International
open.access
The recurrent emergence of drug resistance in Plasmodium falciparum increases the urgency to genetically validate drug resistance mechanisms and identify new targets. Reverse genetics have facilitated genome-scale knockout screens in Plasmodium berghei and Toxoplasma gondii, in which pooled transfections of multiple vectors were critical to increasing scale and throughput. These approaches have not yet been implemented in human malaria species such as P. falciparum and P. knowlesi, in part because the extent to which pooled transfections can be performed in these species remains to be evaluated. Here we use next-generation sequencing to quantitate uptake of a pool of 94 barcoded vectors. The distribution of vector acquisition allowed us to estimate the number of barcodes and DNA molecules taken up by the parasite population. Dilution cloning of P. falciparum transfectants showed that individual clones possess as many as seven episomal barcodes, revealing that an intake of multiple vectors is a frequent event despite the inefficient transfection efficiency. Transfection of three spectrally-distinct fluorescent reporters allowed us to evaluate different transfection methods and revealed that schizont-stage transfection limited the tendency for parasites to take up multiple vectors. In contrast to P. falciparum, we observed that the higher transfection efficiency of P. knowlesi resulted in near complete representation of the library. These findings have important implications for how reverse genetics can be scaled in culturable Plasmodium species.