| Recombination-Mediated Genetic Engineering of Plasmodium berghei DNA. | |
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MedLine Citation:
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PMID: 22990774 Owner: NLM Status: In-Data-Review |
Abstract/OtherAbstract:
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DNA of Plasmodium berghei is difficult to manipulate in Escherichia coli by conventional restriction and ligation methods due to its high content of adenine and thymine (AT) nucleotides. This limits our ability to clone large genes and to generate complex vectors for modifying the parasite genome. We here describe a protocol for using lambda Red recombinase to modify inserts of a P. berghei genomic DNA library constructed in a linear, low-copy, phage-derived vector. The method uses primer extensions of 50 bp, which provide sufficient homology for an antibiotic resistance marker to recombine efficiently with a P. berghei genomic DNA insert in E. coli. In a subsequent in vitro Gateway reaction the bacterial marker is replaced with a cassette for selection in P. berghei. The insert is then released and used for transfection. The basic techniques we describe here can be adapted to generate highly efficient vectors for gene deletion, tagging, targeted mutagenesis, or genetic complementation with larger genomic regions. |
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Authors:
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Claudia Pfander; Burcu Anar; Mathieu Brochet; Julian C Rayner; Oliver Billker |
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Publication Detail:
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Type: Journal Article |
Journal Detail:
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Title: Methods in molecular biology (Clifton, N.J.) Volume: 923 ISSN: 1940-6029 ISO Abbreviation: Methods Mol. Biol. Publication Date: 2013 |
Date Detail:
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Created Date: 2012-09-19 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 9214969 Medline TA: Methods Mol Biol Country: United States |
Other Details:
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Languages: eng Pagination: 127-38 Citation Subset: IM |
Affiliation:
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Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK. |
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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