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A genome-scale vector resource enables high-throughput reverse genetic screening in a malaria parasite.

Gomes AR, Bushell E, Schwach F, Girling G, Anar B, Quail MA, Herd C, Pfander C, Modrzynska K, Rayner JC, Billker O - Cell Host Microbe (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Sanger Institute, Hinxton Cambridge CB10 1SA, UK.

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Fitness Measurements Obtained with PlasmoGEM Vectors Targeting Protein Kinases(A) Distribution plot generated from a ranked list of day 7 fitness values measured in triplicate for each of 42 genes in experiment 1 (left axis). The relative abundance of a targeting vector in the electroporation cuvette at the moment of transfection (gray crosses, right axis) did not predict whether a mutant could be obtained. See Figure S2 for relative abundance data of a representative replicate experiment.(B) Fitness of reference mutants averages 1 by definition. Error bars show standard errors (n = 6).(C) Fitness of selected mutants. Error bars as in (B). Asterisk, different from reference mutants as determined by a two-sided t test corrected for multiple testing (p < 0.01; n = 6).
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fig4: Fitness Measurements Obtained with PlasmoGEM Vectors Targeting Protein Kinases(A) Distribution plot generated from a ranked list of day 7 fitness values measured in triplicate for each of 42 genes in experiment 1 (left axis). The relative abundance of a targeting vector in the electroporation cuvette at the moment of transfection (gray crosses, right axis) did not predict whether a mutant could be obtained. See Figure S2 for relative abundance data of a representative replicate experiment.(B) Fitness of reference mutants averages 1 by definition. Error bars show standard errors (n = 6).(C) Fitness of selected mutants. Error bars as in (B). Asterisk, different from reference mutants as determined by a two-sided t test corrected for multiple testing (p < 0.01; n = 6).

Mentions: To analyze growth curves derived from barcode counting we considered two parameters: (1) the relative abundance of each barcode within the pool, and (2) the relative fitness of each mutant, i.e., the rate at which its abundance changed each day. As expected, the four barcodes corresponding to control genes redundant for asexual development replicated rapidly. These were taken to represent wild-type growth (fitness w = 1). Relative abundance and growth rates were both highly reproducible for each barcode between technical and biological replicates (Figures 3B and 3C). We propose that the shape of a growth curve provides a quantitative measure for the fitness of a mutant. In contrast, the relative abundance of a mutant within a pool we consider less informative, since it may be influenced by a number of additional factors, such as the abundance of a vector in the transfection cocktail, the length of its homology arms, or any local variation in recombination rates. Plotting day 7 fitness from a ranked list (Figure 4A) showed that while the relative abundance of vectors in the transfection cocktail varied by up to one order of magnitude (Figure 4A, right axis), this was not a predictor of fitness of the resulting mutants. Fitness is therefore driven by growth rate, not by the amount of a given vector in the starting pool. The attenuating control vectors were associated with a measurable reduction in parasite fitness to between 0.60 and 0.73, as expected. The majority of protein kinase mutants either had wild-type fitness or were not detected (w = 0). While all reference barcodes robustly replicated close to the average fitness of 1.0 (Figure 4B), the attenuating vectors and some kinase mutants showed statistically significant reductions in fitness that were consistently measured across different days of the infection (Figure 4C). Taken together, these data strongly suggest that barcode counting can be used to phenotype large numbers of mutants in parallel.


A genome-scale vector resource enables high-throughput reverse genetic screening in a malaria parasite.

Gomes AR, Bushell E, Schwach F, Girling G, Anar B, Quail MA, Herd C, Pfander C, Modrzynska K, Rayner JC, Billker O - Cell Host Microbe (2015)

Fitness Measurements Obtained with PlasmoGEM Vectors Targeting Protein Kinases(A) Distribution plot generated from a ranked list of day 7 fitness values measured in triplicate for each of 42 genes in experiment 1 (left axis). The relative abundance of a targeting vector in the electroporation cuvette at the moment of transfection (gray crosses, right axis) did not predict whether a mutant could be obtained. See Figure S2 for relative abundance data of a representative replicate experiment.(B) Fitness of reference mutants averages 1 by definition. Error bars show standard errors (n = 6).(C) Fitness of selected mutants. Error bars as in (B). Asterisk, different from reference mutants as determined by a two-sided t test corrected for multiple testing (p < 0.01; n = 6).
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362957&req=5

fig4: Fitness Measurements Obtained with PlasmoGEM Vectors Targeting Protein Kinases(A) Distribution plot generated from a ranked list of day 7 fitness values measured in triplicate for each of 42 genes in experiment 1 (left axis). The relative abundance of a targeting vector in the electroporation cuvette at the moment of transfection (gray crosses, right axis) did not predict whether a mutant could be obtained. See Figure S2 for relative abundance data of a representative replicate experiment.(B) Fitness of reference mutants averages 1 by definition. Error bars show standard errors (n = 6).(C) Fitness of selected mutants. Error bars as in (B). Asterisk, different from reference mutants as determined by a two-sided t test corrected for multiple testing (p < 0.01; n = 6).
Mentions: To analyze growth curves derived from barcode counting we considered two parameters: (1) the relative abundance of each barcode within the pool, and (2) the relative fitness of each mutant, i.e., the rate at which its abundance changed each day. As expected, the four barcodes corresponding to control genes redundant for asexual development replicated rapidly. These were taken to represent wild-type growth (fitness w = 1). Relative abundance and growth rates were both highly reproducible for each barcode between technical and biological replicates (Figures 3B and 3C). We propose that the shape of a growth curve provides a quantitative measure for the fitness of a mutant. In contrast, the relative abundance of a mutant within a pool we consider less informative, since it may be influenced by a number of additional factors, such as the abundance of a vector in the transfection cocktail, the length of its homology arms, or any local variation in recombination rates. Plotting day 7 fitness from a ranked list (Figure 4A) showed that while the relative abundance of vectors in the transfection cocktail varied by up to one order of magnitude (Figure 4A, right axis), this was not a predictor of fitness of the resulting mutants. Fitness is therefore driven by growth rate, not by the amount of a given vector in the starting pool. The attenuating control vectors were associated with a measurable reduction in parasite fitness to between 0.60 and 0.73, as expected. The majority of protein kinase mutants either had wild-type fitness or were not detected (w = 0). While all reference barcodes robustly replicated close to the average fitness of 1.0 (Figure 4B), the attenuating vectors and some kinase mutants showed statistically significant reductions in fitness that were consistently measured across different days of the infection (Figure 4C). Taken together, these data strongly suggest that barcode counting can be used to phenotype large numbers of mutants in parallel.

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.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Sanger Institute, Hinxton Cambridge CB10 1SA, UK.

Show MeSH
Related in: MedlinePlus