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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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Absence of Evidence for Multiple Integration Events(A) Vector pools were transfected into marker-free lines with pre-existing barcodes in cdpk1, cdpk3, or cdpk4. New barcodes account for approximately half of the total, as would be expected if each parasite genome carried exactly one new barcode. The slight overrepresentation of background barcodes on day 4 probably comes from parasites that failed to integrate a vector and which were not yet completely eliminated after only 3 days of drug selection. All data points are supported by three experiments, and error bars show standard deviations. See Figure S3 for genotyping of marker-free lines.(B) PCR genotyping was performed on parasite gDNA from six infected mice, each transfected with one of three final targeting vectors in the presence of a 20-fold excess of intermediate vectors (10 μg total DNA per transfection), which have the same homology arms but a zeocin resistance cassette that cannot be selected in P. berghei. Presence of intermediate vectors in the input cocktail but absence in the resistant parasite populations suggests that multiple integration events are rare or absent, since hitchhiking of marker free insertions would otherwise be observed.
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fig6: Absence of Evidence for Multiple Integration Events(A) Vector pools were transfected into marker-free lines with pre-existing barcodes in cdpk1, cdpk3, or cdpk4. New barcodes account for approximately half of the total, as would be expected if each parasite genome carried exactly one new barcode. The slight overrepresentation of background barcodes on day 4 probably comes from parasites that failed to integrate a vector and which were not yet completely eliminated after only 3 days of drug selection. All data points are supported by three experiments, and error bars show standard deviations. See Figure S3 for genotyping of marker-free lines.(B) PCR genotyping was performed on parasite gDNA from six infected mice, each transfected with one of three final targeting vectors in the presence of a 20-fold excess of intermediate vectors (10 μg total DNA per transfection), which have the same homology arms but a zeocin resistance cassette that cannot be selected in P. berghei. Presence of intermediate vectors in the input cocktail but absence in the resistant parasite populations suggests that multiple integration events are rare or absent, since hitchhiking of marker free insertions would otherwise be observed.

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.


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)

Absence of Evidence for Multiple Integration Events(A) Vector pools were transfected into marker-free lines with pre-existing barcodes in cdpk1, cdpk3, or cdpk4. New barcodes account for approximately half of the total, as would be expected if each parasite genome carried exactly one new barcode. The slight overrepresentation of background barcodes on day 4 probably comes from parasites that failed to integrate a vector and which were not yet completely eliminated after only 3 days of drug selection. All data points are supported by three experiments, and error bars show standard deviations. See Figure S3 for genotyping of marker-free lines.(B) PCR genotyping was performed on parasite gDNA from six infected mice, each transfected with one of three final targeting vectors in the presence of a 20-fold excess of intermediate vectors (10 μg total DNA per transfection), which have the same homology arms but a zeocin resistance cassette that cannot be selected in P. berghei. Presence of intermediate vectors in the input cocktail but absence in the resistant parasite populations suggests that multiple integration events are rare or absent, since hitchhiking of marker free insertions would otherwise be observed.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4362957&req=5

fig6: Absence of Evidence for Multiple Integration Events(A) Vector pools were transfected into marker-free lines with pre-existing barcodes in cdpk1, cdpk3, or cdpk4. New barcodes account for approximately half of the total, as would be expected if each parasite genome carried exactly one new barcode. The slight overrepresentation of background barcodes on day 4 probably comes from parasites that failed to integrate a vector and which were not yet completely eliminated after only 3 days of drug selection. All data points are supported by three experiments, and error bars show standard deviations. See Figure S3 for genotyping of marker-free lines.(B) PCR genotyping was performed on parasite gDNA from six infected mice, each transfected with one of three final targeting vectors in the presence of a 20-fold excess of intermediate vectors (10 μg total DNA per transfection), which have the same homology arms but a zeocin resistance cassette that cannot be selected in P. berghei. Presence of intermediate vectors in the input cocktail but absence in the resistant parasite populations suggests that multiple integration events are rare or absent, since hitchhiking of marker free insertions would otherwise be observed.
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.

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