Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis.
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/.
Gong
Shiaoching
S
The Rockefeller University, New York, New York, USA. gongs@rockefeller.edu
Kus
Laura
L
Heintz
Nathaniel
N
D000818:Animals; D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D030541:Databases, Genetic; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry; D015723:Gene Library; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D051379:Mice; D008822:Mice, Transgenic; D011938:Rec A Recombinases / Q000235:genetics; D011995:Recombination, Genetic; D018741:Replication Origin / Q000235:genetics*; D019076:Transgenes
D004269:DNA, Bacterial; D005456:Fluorescent Dyes; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011938:Rec A Recombinases