Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis.

Abstract:

:We report here a high-throughput method for the modification of bacterial artificial chromosomes (BACs) that uses a novel two-plasmid approach. In this protocol, a vector modified in our laboratory to hold an R6Kγ origin of replication and a marker recombination cassette is inserted into a BAC in a single recombination step. Temporal control of recombination is achieved through the use of a second plasmid, pSV1.RecA, which possesses a recombinase gene and a temperature-sensitive origin of replication. This highly efficient protocol has allowed us to successfully modify more than 2,000 BACs, from which over 1,000 BAC transgenic mice have been generated. A complete cycle from BAC choice to embryo implantation takes about 5 weeks. Marker genes introduced into the mice include EGFP and EGFP-L10a. All vectors used in this project can be obtained from us by request, and the EGFP reporter mice are available through the Mutant Mouse Regional Resource Center (NINDS/GENSAT collection). CNS anatomical expression maps of the mice are available to the public at http://www.gensat.org/.

journal_name

Nat Protoc

journal_title

Nature protocols

authors

Gong S,Kus L,Heintz N

doi

10.1038/nprot.2010.131

subject

Has Abstract

pub_date

2010-09-01 00:00:00

pages

1678-96

issue

10

eissn

1754-2189

issn

1750-2799

pii

nprot.2010.131

journal_volume

5

pub_type

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