RRC ID 48832
Author Gong S, Kus L, Heintz N.
Title Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis.
Journal Nat Protoc
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/.
Volume 5(10)
Pages 1678-96
Published 2010-9
DOI 10.1038/nprot.2010.131
PII nprot.2010.131
PMID 20885380
PMC PMC3104474
MeSH Animals Chromosomes, Artificial, Bacterial / genetics* DNA, Bacterial / genetics Databases, Genetic Escherichia coli / genetics Fluorescent Dyes / chemistry Gene Library Gene Transfer Techniques* Genetic Vectors / genetics* Green Fluorescent Proteins / chemistry Mice Mice, Transgenic Rec A Recombinases / genetics Recombination, Genetic Replication Origin / genetics* Transgenes
IF 12.423
Times Cited 32
WOS Category BIOCHEMICAL RESEARCH METHODS
Resource
DNA material MSM Mouse BAC clone (RDB04214)