Biomedical Engineering Reference
In-Depth Information
require only the recombinase and its target sequence.
Of these, the most extensively used are Cre recom-
binase from bacteriophage P1 (Lewanodski & Martin
1997) and FLP recombinase (flippase) from the
2
DNA, leaving a single
loxP
site remaining in the
genome. If the
loxP
sites are arranged as inverted
repeats, the intervening DNA segment is inverted.
Both reactions are reversible. However, when the
loxP
sites are arranged in the same orientation,
excision is favoured over reintegration, because the
excised DNA fragment is rapidly degraded.
The ability of flanking
loxP
sites to delineate any
sequence of interest for site-specific deletion has
numerous applications. The most obvious of these is
the deletion of unwanted sequences, such as marker
genes. This approach has been used, for example, as
a simplified strategy to generate targeted mutant
mice containing point mutations. Recall from Chap-
ter 11 that traditional strategies for generating subtle
mutants in mice involve two rounds of homologous
recombination in embryonic stem cells (ES cells)
(p. 207). Such strategies are very inefficient, because
homologous recombination is a rare event. However,
in the Cre recombinase-based approach, a second
round of homologous recombination is unnecessary
(Kilby
et al.
1993). As shown in Fig. 13.7, gene tar-
geting is used to replace the wild-type allele of a
given endogenous gene with an allele containing a
point mutation, and simultaneously to introduce
markers, such as
neo
and
Tk
, for positive and negative
selection. The positive-negative selection markers
within the homology region are flanked by
loxP
sites.
m plasmid of the yeast
Saccharomyces cerevisiae
(Buchholz
et al.
1998). These have been shown to
function in many heterologous eukaryotic systems
including mammalian cells and transgenic animals
and plants (reviewed by Sauer 1994, Ow 1996,
Metzger & Feil 1999). Both recombinases recognize
34 bp sites (termed
loxP
and
FRP
, respectively) com-
prising a pair of 13 bp inverted repeats surrounding
an 8 bp central element.
FRP
possesses an additional
copy of the 13 bp repeat sequence, although this has
been shown to be non-essential for recombination.
Cre recombinase has been used most extensively in
mammals, because it works optimally at 37°C. The
optimal temperature for FLP recombinase is 30°C
(Buchholz
et al.
1996).
µ
Site-specific deletion of transgene
sequences
Site-specific deletion of unwanted transgenes
The reaction catalysed by Cre recombinase is shown
in Fig. 13.6. If two
loxP
sites are arranged as direct
repeats, the recombinase will delete any intervening
A
T
T
A
A
T
A
T
C
G
T
A
T
A
C
G
G
C
T
A
A
T
T
A
A
T
A
T
T
A
G
C
T
A
A
T
T
A
G
C
C
G
T
A
A
T
T
A
A
T
C
G
G
C
A
T
A
T
G
C
T
A
T
A
A
T
T
A
CRE
+
CRE
Fig. 13.6
Structure of the
loxP
site and reactions
catalysed by Cre recombinase when paired
loxP
sites, shown as arrows, are arranged in different
orientations.
