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

TitleRapid bacterial artificial chromosome modification for large-scale mouse transgenesis.
Publication TypeJournal Article
Year of Publication2010
AuthorsGong S, Kus L, Heintz N
JournalNat Protoc
Date Published2010 Sep
KeywordsAnimals, Chromosomes, Artificial, Bacterial, Databases, Genetic, DNA, Bacterial, Escherichia coli, Fluorescent Dyes, Gene Library, Gene Transfer Techniques, Genetic Vectors, Green Fluorescent Proteins, Mice, Mice, Transgenic, Rec A Recombinases, Recombination, Genetic, Replication Origin, Transgenes

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/.

Alternate JournalNat Protoc
PubMed ID20885380
PubMed Central IDPMC3104474
Grant List / / Howard Hughes Medical Institute / United States
N01 NS072370 / NS / NINDS NIH HHS / United States
N01 NS-7-2370 / NS / NINDS NIH HHS / United States