Simple and straightforward construction of a mouse gene targeting vector using in vitro transposition reactions

In a gene targeting experiment, the generation of a targeting construct often requires complex DNA manipulations. We developed a set of cassettes and plasmids useful for creating targeting vectors to modify the mammalian genome. A positive selection marker cassette (PGK/EM7p-npt), which included dua...

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Bibliographic Details
Published in:Nucleic acids research 2005-01, Vol.33 (5), p.e52-e52
Main Authors: Aoyama, Minako, Agari, Kazuko, Sun-Wada, Ge-Hong, Futai, Masamitsu, Wada, Yoh
Format: Article
Language:English
Subjects:
Alleles
Animals
Base Sequence
Bleomycin - pharmacology
DNA Transposable Elements
Escherichia coli - drug effects
Escherichia coli - genetics
Gene Targeting - methods
Genetic Vectors - chemistry
Gentamicins - pharmacology
Kanamycin Resistance
Methods Online
Mice - genetics
Molecular Sequence Data
Plasmids - chemistry
Promoter Regions, Genetic
Recombination, Genetic
Vesicular Transport Proteins - genetics
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Summary:In a gene targeting experiment, the generation of a targeting construct often requires complex DNA manipulations. We developed a set of cassettes and plasmids useful for creating targeting vectors to modify the mammalian genome. A positive selection marker cassette (PGK/EM7p-npt), which included dual prokaryotic and eukaryotic promoters to permit consecutive selection for recombination in Escherichia coli and then in mouse embryonic stem cells, was flanked by two FRT-loxP sequences. The PGK/EM7p-npt cassette was placed between the minimum regions of a Tn7 transposable element for insertion into another DNA by means of Tn7 transposase in vitro. We also constructed a plasmid having a loxP-Zeo-loxP cassette between the modified Tn5 outer elements. These cassettes can be integrated randomly into a given genomic DNA through the in vitro transposition reaction, thus producing a collection of genomic segments flanked by loxP sites (floxed) at various positions without the use of restriction enzymes and DNA ligase. We confirmed that this system remarkably reduced the time and labor for the construction of complex gene targeting vectors.
ISSN:0305-1048
1362-4962
1362-4962
DOI:10.1093/nar/gni055