publication . Article . Other literature type . 2011

A scalable pipeline for highly effective genetic modification of a malaria parasite

Pfander, Claudia; Anar, Burcu; Schwach, Frank; Otto, Thomas D; Brochet, Mathieu; Volkmann, Katrin; Quail, Michael A; Pain, Arnab; Rosen, Barry; Skarnes, William; ...
Open Access
  • Published: 23 Oct 2011 Journal: Nature Methods, volume 8, pages 1,078-1,082 (issn: 1548-7091, eissn: 1548-7105, Copyright policy)
  • Publisher: Springer Nature
Abstract
In malaria parasites the systematic experimental validation of drug and vaccine targets by reverse genetics is constrained by the inefficiency of homologous recombination and by the difficulty of manipulating adenine and thymine (AT) rich Plasmodium DNA in E. coli. We overcome these roadblocks by demonstrating that a high integrity library of P. berghei genomic DNA (>77% AT) in a bacteriophage N15-based vector can be modified efficiently using the lambda Red method of recombineering. We built a pipeline for generating Plasmodium berghei genetic modification vectors at genome scale in serial liquid cultures on 96-well plates. Vectors have long homology arms, whic...
Subjects
Medical Subject Headings: parasitic diseases
free text keywords: Biotechnology, Cell Biology, Biochemistry, Molecular Biology, DNA Modification, Gene Modification, Biology, Genetics, Plasmodium berghei, biology.organism_classification, Recombineering, Malaria, medicine.disease, medicine, Genome, Parasite hosting, Bioinformatics, Article
Funded by
RCUK| A systematic analysis of protein kinases in malaria parasites to identify, validate and prioritise novel drug targets
Project
  • Funder: Research Council UK (RCUK)
  • Project Code: G0501670
  • Funding stream: MRC
,
WT| Experimental genetics of malaria parasites and parasite-host interactions.
Project
  • Funder: Wellcome Trust (WT)
  • Project Code: 089085
  • Funding stream: Cellular and Molecular Neuroscience
,
EC| EVIMALAR
Project
EVIMALAR
Towards the establishment of a permanent European Virtual Institute dedicated to Malaria Research (EVIMalaR).
  • Funder: European Commission (EC)
  • Project Code: 242095
  • Funding stream: FP7 | SP1 | HEALTH
33 references, page 1 of 3

Janse, CJ, Franke-Fayard, B, Waters, AP. Selection by flow-sorting of genetically transformed, GFP-expressing blood stages of the rodent malaria parasite, Plasmodium berghei. Nat Protoc. 2006; 1: 614-623 [PubMed]

Rutherford, K. Artemis: sequence visualization and annotation. Bioinformatics. 2000; 16: 944-945 [OpenAIRE] [PubMed]

Zerbino, DR, Birney, E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008; 18: 821-829 [OpenAIRE] [PubMed]

Assefa, S, Keane, TM, Otto, TD, Newbold, C, Berriman, M. ABACAS: algorithm-based automatic contiguation of assembled sequences. Bioinformatics. 2009; 25: 1968-1969 [OpenAIRE] [PubMed]

Li, H. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25: 2078-2079 [OpenAIRE] [PubMed]

Janse, CJ. High efficiency transfection of Plasmodium berghei facilitates novel selection procedures. Mol Biochem Parasitol. 2006; 145: 60-70 [PubMed]

Crabb, BS, Cowman, AF. Characterization of promoters and stable transfection by homologous and nonhomologous recombination in Plasmodium falciparum. Proc Natl Acad Sci U S A. 1996; 93: 7289-7294 [OpenAIRE] [PubMed]

van Dijk, MR, Janse, CJ, Waters, AP. Expression of a Plasmodium gene introduced into subtelomeric regions of Plasmodium berghei chromosomes. Science. 1996; 271: 662-665 [OpenAIRE] [PubMed]

Wu, Y, Kirkman, LA, Wellems, TE. Transformation of Plasmodium falciparum malaria parasites by homologous integration of plasmids that confer resistance to pyrimethamine. Proc Natl Acad Sci U S A. 1996; 93: 1130-1134 [OpenAIRE] [PubMed]

Tewari, R. The systematic functional analysis of Plasmodium protein kinases identifies essential regulators of mosquito transmission. Cell Host Microbe. 2010; 8: 377-387 [OpenAIRE] [PubMed]

Maier, AG. Exported proteins required for virulence and rigidity of Plasmodium falciparum-infected human erythrocytes. Cell. 2008; 134: 48-61 [OpenAIRE] [PubMed]

Janse, CJ, Ramesar, J, Waters, AP. High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei. Nat Protoc. 2006; 1: 346-356 [OpenAIRE] [PubMed]

Hasty, P, Rivera-Perez, J, Bradley, A. The length of homology required for gene targeting in embryonic stem cells. Mol Cell Biol. 1991; 11: 5586-5591 [OpenAIRE] [PubMed]

Shulman, MJ, Nissen, L, Collins, C. Homologous recombination in hybridoma cells: dependence on time and fragment length. Mol Cell Biol. 1990; 10: 4466-4472 [OpenAIRE] [PubMed]

Gardner, MJ. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature. 2002; 419: 498-511 [OpenAIRE] [PubMed]

33 references, page 1 of 3
Abstract
In malaria parasites the systematic experimental validation of drug and vaccine targets by reverse genetics is constrained by the inefficiency of homologous recombination and by the difficulty of manipulating adenine and thymine (AT) rich Plasmodium DNA in E. coli. We overcome these roadblocks by demonstrating that a high integrity library of P. berghei genomic DNA (>77% AT) in a bacteriophage N15-based vector can be modified efficiently using the lambda Red method of recombineering. We built a pipeline for generating Plasmodium berghei genetic modification vectors at genome scale in serial liquid cultures on 96-well plates. Vectors have long homology arms, whic...
Subjects
Medical Subject Headings: parasitic diseases
free text keywords: Biotechnology, Cell Biology, Biochemistry, Molecular Biology, DNA Modification, Gene Modification, Biology, Genetics, Plasmodium berghei, biology.organism_classification, Recombineering, Malaria, medicine.disease, medicine, Genome, Parasite hosting, Bioinformatics, Article
Funded by
RCUK| A systematic analysis of protein kinases in malaria parasites to identify, validate and prioritise novel drug targets
Project
  • Funder: Research Council UK (RCUK)
  • Project Code: G0501670
  • Funding stream: MRC
,
WT| Experimental genetics of malaria parasites and parasite-host interactions.
Project
  • Funder: Wellcome Trust (WT)
  • Project Code: 089085
  • Funding stream: Cellular and Molecular Neuroscience
,
EC| EVIMALAR
Project
EVIMALAR
Towards the establishment of a permanent European Virtual Institute dedicated to Malaria Research (EVIMalaR).
  • Funder: European Commission (EC)
  • Project Code: 242095
  • Funding stream: FP7 | SP1 | HEALTH
33 references, page 1 of 3

Janse, CJ, Franke-Fayard, B, Waters, AP. Selection by flow-sorting of genetically transformed, GFP-expressing blood stages of the rodent malaria parasite, Plasmodium berghei. Nat Protoc. 2006; 1: 614-623 [PubMed]

Rutherford, K. Artemis: sequence visualization and annotation. Bioinformatics. 2000; 16: 944-945 [OpenAIRE] [PubMed]

Zerbino, DR, Birney, E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 2008; 18: 821-829 [OpenAIRE] [PubMed]

Assefa, S, Keane, TM, Otto, TD, Newbold, C, Berriman, M. ABACAS: algorithm-based automatic contiguation of assembled sequences. Bioinformatics. 2009; 25: 1968-1969 [OpenAIRE] [PubMed]

Li, H. The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25: 2078-2079 [OpenAIRE] [PubMed]

Janse, CJ. High efficiency transfection of Plasmodium berghei facilitates novel selection procedures. Mol Biochem Parasitol. 2006; 145: 60-70 [PubMed]

Crabb, BS, Cowman, AF. Characterization of promoters and stable transfection by homologous and nonhomologous recombination in Plasmodium falciparum. Proc Natl Acad Sci U S A. 1996; 93: 7289-7294 [OpenAIRE] [PubMed]

van Dijk, MR, Janse, CJ, Waters, AP. Expression of a Plasmodium gene introduced into subtelomeric regions of Plasmodium berghei chromosomes. Science. 1996; 271: 662-665 [OpenAIRE] [PubMed]

Wu, Y, Kirkman, LA, Wellems, TE. Transformation of Plasmodium falciparum malaria parasites by homologous integration of plasmids that confer resistance to pyrimethamine. Proc Natl Acad Sci U S A. 1996; 93: 1130-1134 [OpenAIRE] [PubMed]

Tewari, R. The systematic functional analysis of Plasmodium protein kinases identifies essential regulators of mosquito transmission. Cell Host Microbe. 2010; 8: 377-387 [OpenAIRE] [PubMed]

Maier, AG. Exported proteins required for virulence and rigidity of Plasmodium falciparum-infected human erythrocytes. Cell. 2008; 134: 48-61 [OpenAIRE] [PubMed]

Janse, CJ, Ramesar, J, Waters, AP. High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei. Nat Protoc. 2006; 1: 346-356 [OpenAIRE] [PubMed]

Hasty, P, Rivera-Perez, J, Bradley, A. The length of homology required for gene targeting in embryonic stem cells. Mol Cell Biol. 1991; 11: 5586-5591 [OpenAIRE] [PubMed]

Shulman, MJ, Nissen, L, Collins, C. Homologous recombination in hybridoma cells: dependence on time and fragment length. Mol Cell Biol. 1990; 10: 4466-4472 [OpenAIRE] [PubMed]

Gardner, MJ. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature. 2002; 419: 498-511 [OpenAIRE] [PubMed]

33 references, page 1 of 3
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