Advances in transgenic technology - Principles of Gene Manipulation - page 258

This strategy was first used in transgenic mice to
study the effect of SV40 T-antigen expression in
development (Pichel et al. 1993). In this case, Cre
recombinase was expressed under the control of a
developmentally regulated promoter. Essentially the
same strategy was used in transgenic tobacco plants
to activate a reporter gene in seeds (Odell et al.
1994). In this case, Cre recombinase was expressed
under the control of a seed-specific promoter. An
important feature of both these experiments was the
use of two separate transgenic lines, one expressing
Cre recombinase in a regulated manner and one
containing the target gene. Crosses between these
lines brought both transgenes together in the hybrid
progeny, resulting in the conditional activation of
the transgene based on the expression profile of
Cre. This is an extremely versatile and widely used
strategy, because it allows 'mix and match' between
different Cre transgenic and 'responder' lines. We
return to this subject below.

nature of site-specific recombination, transgenic loci
generated by this method are likely to be less com-
plex than loci generated by random integration.

Chromosome engineering

Site-specific recombination between widely separated
target sites or target sites on different chromosomes
can be used to generate large deletions, translocations
and other types of chromosome mutation. Chromo-
some engineering by site-specific recombination
was first reported by Golic (1991), using FLP recom-
binase in Drosophila , but similar experiments have
now been carried out in plants and mice. Precise intra-
chromosomal deletions can be generated in mice by
two rounds of gene targeting, introducing loxP sites
at distant sites, followed by Cre-mediated recombin-
ation (Ramirez-Solis et al. 1995, Li et al. 1996). In
plants, where gene targeting is very inefficient, an
ingenious scheme has been developed where loxP
sites are arranged in tandem on a transformation
construct, one inside a Ds transposon and one out-
side (Fig. 13.9). The transposon is placed between a
marker gene and its promoter. When this construct is
introduced into tobacco plants containing the auto-
nomous transposon Ac to provide a source of trans-
posase, the Ds element can excise from the transgene,
as revealed by marker-gene expression. In most
heterologous plants, Ac-Ds elements reintegrate at a
position that is linked to the original site. Although
the site of reintegration cannot be controlled, this
nevertheless defines a large chromosomal segment
that can be excised by Cre recombinase (Medberry
et al. 1995, Osbourne et al. 1995). Translocations
are more difficult to engineer, because interchromo-
somal site-specific recombination is inefficient, and
inventive selection strategies are required to identify
the desired products (e.g. see Qin et al. 1994, Smith
et al. 1995, Van Deursen et al. 1995).

Site-specific transgene integration

Site-specific integration of transgenes can occur if
the genome contains a recombinase recognition
site. This may be introduced by random integration
or (in mice) by gene targeting. Using an unmodified
Cre- loxP system, transgene integration occurs at a
low efficiency, because, as discussed above, the equi-
librium of the reaction is shifted in favour of excision.
Initial attempts to overcome this problem by provid-
ing transient Cre activity had limited success (see
Sauer & Henderson 1990, Baubonis & Saur 1993).
However, high-efficiency Cre-mediated integration
has been achieved in plants (Albert et al. 1995) and
mammalian cells (Feng et al. 1999) using mutated
or inverted loxP sites. Site-specific transgene integ-
ration into mammalian cells has also been achieved
using FLP recombinase (O'Gorman et al. 1991).
Transgene integration by site-specific recom-
bination has many advantages over the random
integration that is normally achieved by illegitimate
recombination. For example, if a region of the genome
can be identified that is not subject to negative
position effects (Box 11.1), transgenic lines with a
loxP site at this position can be used for the stable
and high-level expression of any transgene (e.g.
Fukushige & Sauer 1992). Also, due to the precise

Cre-mediated conditional
mutants in mice

In mice, gene targeting and site-specific recombina-
tion can be used in a powerful combined approach to
generate conditional knockout mutants. Essentially,
targeting vectors are designed so that part of a
selected endogenous gene becomes flanked by loxP