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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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PlasmoGEM: A Genome Scale Free Resource of Genetic Modification Vectors for P. berghei Reverse Genetics(A) A diagram of the modular vector production process showing the efficiency at each step (red), as well as resources (gray boxes) and data (dashed lines) submitted to the database.(B) Genome coverage achieved to date.(C) Schematic showing knockout vector designs and locations of the gene-specific molecular barcode included in each vector.(D) Default C-terminal epitope-tagging vector and a panel of alternative fusion tags.
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fig1: PlasmoGEM: A Genome Scale Free Resource of Genetic Modification Vectors for P. berghei Reverse Genetics(A) A diagram of the modular vector production process showing the efficiency at each step (red), as well as resources (gray boxes) and data (dashed lines) submitted to the database.(B) Genome coverage achieved to date.(C) Schematic showing knockout vector designs and locations of the gene-specific molecular barcode included in each vector.(D) Default C-terminal epitope-tagging vector and a panel of alternative fusion tags.

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).


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)

PlasmoGEM: A Genome Scale Free Resource of Genetic Modification Vectors for P. berghei Reverse Genetics(A) A diagram of the modular vector production process showing the efficiency at each step (red), as well as resources (gray boxes) and data (dashed lines) submitted to the database.(B) Genome coverage achieved to date.(C) Schematic showing knockout vector designs and locations of the gene-specific molecular barcode included in each vector.(D) Default C-terminal epitope-tagging vector and a panel of alternative fusion tags.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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

fig1: PlasmoGEM: A Genome Scale Free Resource of Genetic Modification Vectors for P. berghei Reverse Genetics(A) A diagram of the modular vector production process showing the efficiency at each step (red), as well as resources (gray boxes) and data (dashed lines) submitted to the database.(B) Genome coverage achieved to date.(C) Schematic showing knockout vector designs and locations of the gene-specific molecular barcode included in each vector.(D) Default C-terminal epitope-tagging vector and a panel of alternative fusion tags.
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).

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