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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Aoyama M,Agari K,Sun-Wada GH,Futai M,Wada Y

doi

10.1093/nar/gni055

keywords:

subject

Has Abstract

pub_date

2005-03-22 00:00:00

pages

e52

issue

5

eissn

0305-1048

issn

1362-4962

pii

33/5/e52

journal_volume

33

pub_type

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