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Nat Protoc. 2010 Sep;5(10):1678-96. doi: 10.1038/nprot.2010.131. Epub 2010 Sep 30.

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

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1
The Rockefeller University, New York, New York, USA. gongs@rockefeller.edu

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/.

PMID:
20885380
PMCID:
PMC3104474
DOI:
10.1038/nprot.2010.131
[Indexed for MEDLINE]
Free PMC Article
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