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: Finally we asked whether the interpretation of barcode counting data from parallel transfection experiments could be confounded by multiple vectors integrating into the same parasite genome. First we transfected pools of barcoded vectors into three parasite clones, each of which already carried a different barcode from the insertion of a targeting vector and subsequent recycling of the selection marker. We reasoned that if each mutant integrated one new vector, the pre-existing barcode should account for exactly half of all barcode counts after transfection and drug selection. The data were entirely consistent with this model (Figure 6A), suggesting that if multiple integration events existed, they would be too rare to be isolated by limiting dilution cloning. To detect rare double integration events, we chose three genes that are readily disrupted using PlasmoGEM vectors. For each we transfected the final knockout vector together with a 20-fold excess of the selection marker-free intermediate vectors for the other two genes. We expected to detect replication of the marker-free vectors by PCR, but only if their genomic integration coincided with integration of the selectable construct into the same genome. These experiments failed to generate evidence for multiple integration events into the same genome (Figure 6B). The data indicate that different homologous integration events in P. berghei are independent, and suggest that DNA uptake after electroporation is not the factor limiting transfection efficiency.
Affiliation: Wellcome Trust Sanger Institute, Hinxton Cambridge CB10 1SA, UK.