Title | Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis. |
Publication Type | Journal Article |
Year of Publication | 2010 |
Authors | Gong S, Kus L, Heintz N |
Journal | Nat Protoc |
Volume | 5 |
Issue | 10 |
Pagination | 1678-96 |
Date Published | 2010 Sep |
ISSN | 1750-2799 |
Keywords | Animals, 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 |
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/. |
DOI | 10.1038/nprot.2010.131 |
Alternate Journal | Nat Protoc |
PubMed ID | 20885380 |
PubMed Central ID | PMC3104474 |
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 |