Plant Genome II Conference
Town & Country Conference Center, San Diego, CA, January, 1994.
PG-II: DNA TRANSFER VIA CRE-LOX MEDIATED SITE-SPECIFIC RECOMBINATION
DNA TRANSFER VIA CRE-LOX MEDIATED SITE-SPECIFIC RECOMBINATION.
David W. Ow, Henrik Albert, Elsa Lee and Emily Dale, Plant Gene
Expression Center, USDA-UC Berkeley, Albany, CA 94710
The precise, selective and reproducible placement of DNA
constructs into the plant genome is desirable for the analysis of
transgenes free of variables associated with random-site
integration. We have tested the Cre-lox site-specific
recombination system, where Cre is a 38.5 kDa recombinase and lox
is a 34 bp recognition site, for integrating circular DNA
molecules into lox sites previously placed into the plant genome.
Mutant lox sites that reduce the effectiveness of the reverse
(excision) reaction were compared with wild-type sites for their
ability to allow DNA integration. Two experimental strategies
were used to abolish Cre activity upon site-specific insertion.
In the first strategy, integration events were recovered only
when mutant lox sites were used. In a second strategy, site-
specific integration events were recovered with either wild-type
or mutant lox sequences. Most integration events were single
copy insertions of the input plasmid at the genomic lox site.
Additional insertions elsewhere in the genome were rarely
observed. Some of the integration events were accompanied by a
rearrangement of the input plasmid; this occurred frequently with
the use of wild-type lox sites. The mutant lox sites, as part of
a Cre-lox mediated gene integration system is an effective tool
for the precise and reproducible insertion of single, circular
DNA molecules into genomic targets. Other recombinase-mediated
gene transfer possibilities will be discussed.
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