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PMID: 12466304 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Highly efficient modification of bacterial artificial chromosomes (BACs) using novel shuttle vectors containing the R6Kgamma origin of replication.

Genome research ·Vol. 12 ·No. 12 ·2002-12-00 ·Pages 1992-8

Gong S, Yang XW, Li C, Heintz N

Abstract

Bacterial artificial chromosome (BAC) mediated transgenesis has proven to be a highly reliable way to obtain accurate transgene expression for in vivo studies of gene expression and function. A rate-limiting step in use of this technology to characterize large numbers of genes has been the process with which BACs can be modified by homologous recombination in Escherichia coli. We report here a highly efficient method for modifying BACs by using a novel set of shuttle vectors that contain the R6Kgamma origin for DNA replication, the E. coli RecA gene for recombination, and the SacB gene for negative selection. These new vectors greatly increased the ease with which one can clone the shuttle vectors, as well as screen for co-integrated and resolved clones. Furthermore, we simplify the shuttle vector cloning to one step by incorporation of a "built-in" resolution cassette for rapid removal of the unwanted vector sequences. This new system has been used to modify a dozen BACs. It is well suited for efficient production of modified BACs for use in a variety of in vivo studies.

MeSH Terms
Animals Chromosomes, Artificial, Bacterial/enzymology,genetics DNA Probes/genetics DNA, Bacterial/genetics Escherichia coli/genetics Gene Library Genetic Vectors/genetics Hexosyltransferases/genetics Humans Mice Mice, Transgenic Mutagenesis, Insertional/methods Rec A Recombinases/genetics Replication Origin/genetics
Chemicals
DNA Probes DNA, Bacterial Hexosyltransferases levansucrase Rec A Recombinases
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Gong Shiaoching
Laboratory of Molecular Biology, The Rockefeller University, New York, New York 10021, USA.
Yang Xiangdong William
Li Chenjian
Heintz Nathaniel
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Article Info
Journal
Genome research
Abbr.
Genome Res
ISSN
1088-9051
DOI
10.1101/gr.476202
Published
2002-12-00
Pages
1992-8
Language
English
Region
United States
NLM ID
9518021
PMCID
PMC187570
Subset
IM
Grants
NINDS NIH HHS · N01-NS0233 · United States
NINDS NIH HHS · NS 39636-03 · United States
External Links
PubMed source DOI: 10.1101/gr.476202 PMC Full Text View journal details
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