A scalable pipeline for highly effective genetic modification of a malaria parasite
| Autor: | Arnab Pain, Claudia Pfander, Michael A. Quail, Frank Schwach, William C. Skarnes, Katrin Volkmann, Julian C. Rayner, Barry Rosen, Mathieu Brochet, Thomas D. Otto, Oliver Billker, Burcu Anar |
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| Rok vydání: | 2011 |
| Předmět: |
Plasmodium berghei
Genetic Vectors DNA Recombinant Biology Biochemistry Genetic recombination Genome Article 03 medical and health sciences 0302 clinical medicine parasitic diseases Escherichia coli medicine Parasite hosting Homologous Recombination Molecular Biology Gene Library 030304 developmental biology Genetics 0303 health sciences Cell Biology DNA Protozoan medicine.disease biology.organism_classification Reverse genetics Malaria 3. Good health Genetic Engineering Homologous recombination Inefficiency Genome Protozoan 030217 neurology & neurosurgery Biotechnology |
| Zdroj: | Nature Methods; Vol 8 Nature methods |
| ISSN: | 1548-7105 1548-7091 |
| Popis: | 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 (A+T)-rich DNA of most Plasmodium species in Escherichia coli. We overcame these roadblocks by creating a high-integrity library of Plasmodium berghei genomic DNA (77% A+T content) in a bacteriophage N15-based vector that can be modified efficiently using the lambda Red method of recombineering. We built a pipeline for generating P. berghei genetic modification vectors at genome scale in serial liquid cultures on 96-well plates. Vectors have long homology arms, which increase recombination frequency up to tenfold over conventional designs. The feasibility of efficient genetic modification at scale will stimulate collaborative, genome-wide knockout and tagging programs for P. berghei. |
| Databáze: | OpenAIRE |
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