A genome-scale vector resource enables high-throughput reverse genetic screening in a malaria parasite.
Bottom Line: We present a large-scale resource of barcoded vectors with long homology arms for effective modification of the Plasmodium berghei genome.To validate the utility of this resource, we rescreen the P. berghei kinome, using published kinome screens for comparison.We find that several protein kinases function redundantly in asexual blood stages and confirm the targetability of kinases cdpk1, gsk3, tkl3, and PBANKA_082960 by genotyping cloned mutants.
Affiliation: Wellcome Trust Sanger Institute, Hinxton Cambridge CB10 1SA, UK.Show MeSH
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Mentions: To generate a genome-scale resource of gene knockout vectors, we used a modular pipeline for recombinase mediated engineering in E. coli (Pfander et al., 2011). The parasite gene of interest was first replaced in appropriately chosen gDNA clones with a marker for positive and negative selection in E. coli using Red/ET recombinase-mediated engineering. The bacterial markers were then exchanged under negative selection for a drug resistance cassette for P. berghei in a single in vitro Gateway recombinase reaction. When applied to the 2,781 P. berghei genes that have any level of functional annotation (57% of the genome), a first pass of the production pipeline yielded gene deletion vectors for 1,868 different protein coding genes of the core nuclear genome (Figures 1A–1C). These vectors form the foundation of the Plasmodium genetic modification resource, PlasmoGEM (Figure 1A), which can be viewed and requested through a searchable database at http://plasmogem.sanger.ac.uk (Schwach et al., 2015).
Affiliation: Wellcome Trust Sanger Institute, Hinxton Cambridge CB10 1SA, UK.