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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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Reproducibility of Independent Barcode Counting Experiment with Respect to the Abundance and Relative Replication Rates of All Barcodes(A) Each experiment involved three replicate transfections of a different schizont culture performed on a different day and using independently prepared vector pools. Error bars show standard errors (n = 3 per experiment). Green lines, four sexual stage genes (p25, p28, p230p 3xHA tag, and soap). Orange lines, three attenuated mutants (plasmepsin IV, PBANKA_110420, PBANKA_140160). Twenty-two mutants are shown in total. See Figure S1 for genotyping data.(B) Linear regression analysis of mean abundance values for the two experiments shown in (B). All barcodes present until day 8 posttransfection were included. Error bars show standard errors of the mean (n = 3).(C) Regression analysis of average mean fitness for each barcode between days 5–8 posttransfection for the two biological replicates in (B). Fitness is calculated from the replication rate of the gene-specific barcode relative to the mean of the four sexual stage reference genes. Error bars show standard errors (n = 3). See Table S1 for fitness measurements for individual vectors, and Table S4, illustrating data analysis.
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fig3: Reproducibility of Independent Barcode Counting Experiment with Respect to the Abundance and Relative Replication Rates of All Barcodes(A) Each experiment involved three replicate transfections of a different schizont culture performed on a different day and using independently prepared vector pools. Error bars show standard errors (n = 3 per experiment). Green lines, four sexual stage genes (p25, p28, p230p 3xHA tag, and soap). Orange lines, three attenuated mutants (plasmepsin IV, PBANKA_110420, PBANKA_140160). Twenty-two mutants are shown in total. See Figure S1 for genotyping data.(B) Linear regression analysis of mean abundance values for the two experiments shown in (B). All barcodes present until day 8 posttransfection were included. Error bars show standard errors of the mean (n = 3).(C) Regression analysis of average mean fitness for each barcode between days 5–8 posttransfection for the two biological replicates in (B). Fitness is calculated from the replication rate of the gene-specific barcode relative to the mean of the four sexual stage reference genes. Error bars show standard errors (n = 3). See Table S1 for fitness measurements for individual vectors, and Table S4, illustrating data analysis.

Mentions: Schizonts cotransfected with a cocktail of 48 vectors and injected into mice (Figure 2) gave rise to drug resistant parasites 4 days later. This was indicative of an overall transfection efficiency of ∼10−4 and suggested that roughly 2,500 independent recombination events occurred in a transfection, enough to generate a complex mixture of mutants. Blood samples were subsequently collected exactly every 24 hr from day 4, gDNA was extracted, and vector-specific barcodes were amplified by a polymerase chain reaction (PCR) with a generic primer pair (see Figure S1A available online) and counted on a benchtop next-generation sequencer (Figure 2). In each of two replicate experiments, the same 22 barcodes from the vector pool were robustly detected and yielded nearly identical growth curves (Figure 3A). Southern hybridization of separated chromosomes showed evidence for vector integration events throughout the genome (Figure S1B). Long-range PCR products confirmed integration events for 17 of the 22 replicating barcodes (Figure S1C).


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)

Reproducibility of Independent Barcode Counting Experiment with Respect to the Abundance and Relative Replication Rates of All Barcodes(A) Each experiment involved three replicate transfections of a different schizont culture performed on a different day and using independently prepared vector pools. Error bars show standard errors (n = 3 per experiment). Green lines, four sexual stage genes (p25, p28, p230p 3xHA tag, and soap). Orange lines, three attenuated mutants (plasmepsin IV, PBANKA_110420, PBANKA_140160). Twenty-two mutants are shown in total. See Figure S1 for genotyping data.(B) Linear regression analysis of mean abundance values for the two experiments shown in (B). All barcodes present until day 8 posttransfection were included. Error bars show standard errors of the mean (n = 3).(C) Regression analysis of average mean fitness for each barcode between days 5–8 posttransfection for the two biological replicates in (B). Fitness is calculated from the replication rate of the gene-specific barcode relative to the mean of the four sexual stage reference genes. Error bars show standard errors (n = 3). See Table S1 for fitness measurements for individual vectors, and Table S4, illustrating data analysis.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig3: Reproducibility of Independent Barcode Counting Experiment with Respect to the Abundance and Relative Replication Rates of All Barcodes(A) Each experiment involved three replicate transfections of a different schizont culture performed on a different day and using independently prepared vector pools. Error bars show standard errors (n = 3 per experiment). Green lines, four sexual stage genes (p25, p28, p230p 3xHA tag, and soap). Orange lines, three attenuated mutants (plasmepsin IV, PBANKA_110420, PBANKA_140160). Twenty-two mutants are shown in total. See Figure S1 for genotyping data.(B) Linear regression analysis of mean abundance values for the two experiments shown in (B). All barcodes present until day 8 posttransfection were included. Error bars show standard errors of the mean (n = 3).(C) Regression analysis of average mean fitness for each barcode between days 5–8 posttransfection for the two biological replicates in (B). Fitness is calculated from the replication rate of the gene-specific barcode relative to the mean of the four sexual stage reference genes. Error bars show standard errors (n = 3). See Table S1 for fitness measurements for individual vectors, and Table S4, illustrating data analysis.
Mentions: Schizonts cotransfected with a cocktail of 48 vectors and injected into mice (Figure 2) gave rise to drug resistant parasites 4 days later. This was indicative of an overall transfection efficiency of ∼10−4 and suggested that roughly 2,500 independent recombination events occurred in a transfection, enough to generate a complex mixture of mutants. Blood samples were subsequently collected exactly every 24 hr from day 4, gDNA was extracted, and vector-specific barcodes were amplified by a polymerase chain reaction (PCR) with a generic primer pair (see Figure S1A available online) and counted on a benchtop next-generation sequencer (Figure 2). In each of two replicate experiments, the same 22 barcodes from the vector pool were robustly detected and yielded nearly identical growth curves (Figure 3A). Southern hybridization of separated chromosomes showed evidence for vector integration events throughout the genome (Figure S1B). Long-range PCR products confirmed integration events for 17 of the 22 replicating barcodes (Figure S1C).

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