title: Simple and straightforward construction of a mouse gene targeting vector using in vitro transposition reactions.
abstract: 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.
authors.affiliation: Division of Biological Science, Institute for Scientific and Industrial Research, Osaka University and CREST, Japan Science and Technology Agency Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan.
authors.forename: Minako
authors.initials: M
authors.lastname: Aoyama
authors.forename: Kazuko
authors.initials: K
authors.lastname: Agari
authors.forename: Ge-Hong
authors.initials: GH
authors.lastname: Sun-Wada
authors.forename: Masamitsu
authors.initials: M
authors.lastname: Futai
authors.forename: Yoh
authors.initials: Y
authors.lastname: Wada
mesh_terms: D000483:Alleles; D000818:Animals; D001483:Base Sequence; D001761:Bleomycin / Q000494:pharmacology; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000737:chemistry*; D005839:Gentamicins / Q000494:pharmacology; D007613:Kanamycin Resistance; D051379:Mice / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D033921:Vesicular Transport Proteins / Q000235:genetics
chemical_list: D004251:DNA Transposable Elements; D005839:Gentamicins; D033921:Vesicular Transport Proteins; D001761:Bleomycin; C105427:Zeocin; C010680:antibiotic G 418