Nucleic Acids Research, Vol 25, Issue 11 2205-2212, Copyright © 1997 by Oxford University Press
PK Chatterjee and JS Coren
A general approach for isolating large nested deletions in P1 artificial
chromosomes (PACs) and bacterial artificial chromosomes (BACs) by
retrofitting with a loxP site-containing Tn10 mini-transposon is described.
Cre-mediated recombination between the loxP site existing in these clones
and one introduced by transposition leads to deletions and inversions of
the DNA between these sites. Large deletions are selectively recovered by
transducing the retrofitted PAC or BAC clones with P1 phage. The
requirement that both loxP sites in the cointegrate be packaged into a P1
head ensures that only large deletions are rescued. PCR analyses identified
these deletions as products of legitimate recombination between loxP sites
mediated by Cre protein. BACs produce deletions much more efficiently than
PACs although the former cannot be induced to greater than unit copy in
cells. Mammalian cell-responsive antibiotic resistance markers are
introduced as part of the transposon into genomic clone deletions for
subsequent functional analysis. Most importantly, the loxP site
retrofitting and P1 transduction can be performed in the same bacterial
host containing these clones directly isolated from PAC or BAC libraries.
These procedures should facilitate physical and functional mapping of genes
and regulatory elements in these large plasmids.
ARTICLES
Isolating large nested deletions in bacterial and P1 artificial chromosomes by in vivo P1 packaging of products of Cre-catalysed recombination between the endogenous and a transposed loxP site
Department of Medicine, SUNY Health Science Center, 750 East Adams Street, Syracuse, NY 13210, USA. chatterp@vax.cs.hscsyr.edu
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