When reverse genetics meets physiology:
the use of site-speci¢c recombinases in mice
Franc ?ois Tronchea;?, Emilio Casanovab, Marc Turiaulta, Iman Sahlya, Christoph Kellendonkc
aCNRS FRE2401, Molecular Genetics, Neurophysiology and Behavior, Institute of Biology, Colle 'ge de France, 11 place Marcelin Berthelot,
75231 Paris Cedex 5, France
bDepartment of Physiology, Biozentrum/Pharmazentrum, University of Basel, Basel, Switzerland
cCenter for Neurobiology and Behaviour, Columbia University, New York, NY, USA
Received 14 August 2002; accepted 16 August 2002
First published online 27 August 2002
Edited by Gunnar von Heijne
cise introduction of de¢ned genetic mutations into the mouse
genome. In theory, any deletion, point mutation, inversion or
translocation can be modeled in mice. Because gene targeting
is controlled both spatially and temporally, the function of a
given gene can be studied in the desired cell types and at a
speci¢c time point. This ‘genetic dissection’ allows to de¢ne
gene function in development, physiology or behavior. In
this review, we focus on the technical possibilities of Cre and
other site-speci¢c recombinases but also discuss their limit-
ations. ? 2002 Published by Elsevier Science B.V. on behalf
of the Federation of European Biochemical Societies.
The use of site-speci¢c recombinases enables the pre-
Key words: Recombinase; Cre-loxP; Mutation;
During the last decade, the development of new genetic
tools revolutionized reverse genetics in the mammalian organ-
ism. The possibility to speci¢cally modify a gene locus by
homologous recombination in mouse embryonic stem (ES)
cells and to observe the consequences of this modi¢cation in
living animals provided invaluable information. However, the
study of the ¢rst generation of mouse mutants was limited by
several technical caveats. First, gene targeting in ES cells re-
quires the use of selection markers, usually an open reading
frame (ORF) encoding the neomycin resistance gene under the
control of a strong promoter. The presence of this promoter
has been shown to in£uence the expression levels of neighbor-
ing genes and of the gene of interest itself. The latter can be a
problem if subtle genetic modi¢cations such as the introduc-
tion of point mutations rather than a total inactivation of the
gene of interest is the aim. Second, in many instances the
inactivation of a gene can be lethal at early embryonic or
postnatal development, preventing the study of its function
at later stages. Third, if a gene is expressed in many di¡erent
cell types, its inactivation may lead to a complex and non-
interpretable phenotype that is caused by the accumulation of
di¡erent alterations in each cell type.
Ten years ago, Lakso et al. and Orban et al. demonstrated
the use of a site-speci¢c recombinase in mice [1,2]. This ap-
proach has revolutionized reverse genetics and its further de-
velopment has already solved some of the described caveats.
2. The Cre-loxP system
The Cre recombinase (Cyclization recombination) is a
member of the integrase family of site-speci¢c recombinases.
This protein family groups more than 100 members found in
Archae, Eubacteria, mitochondria and yeasts . They are
involved in integration and excision of viral and plasmid
DNA, transposition, resolution of catenated DNA circles,
DNA excision and the control of gene expression.
Cre is a 38 kDa protein encoded by the bacteriophage P1
that recognizes a 34 bp DNA target on the P1 genome called
loxP (locus of X-over of P1). It catalyzes reciprocal DNA
recombination between two loxP sites. This mechanism serves
to cyclize P1 DNA after infections and during bacterial divi-
sion, it facilitates the segregation of P1 phages by resolving
dimeric plasmids that were before formed by homologous
recombination . LoxP sites are composed of two inverted
DNA segments of 13 bp Cre monomer binding sites and a
spacer of 8 bp (Fig. 1A). In vitro and crystallographic studies
revealed the molecular mechanisms of Cre-mediated recombi-
nation. Two Cre monomers bind cooperatively to the loxP
site with nanomolar a⁄nity. In the absence of any accessory
factors, two loxP sites are assembled in an antiparallel fashion
by four Cre monomers to form a synaptic structure stabilized
by cyclic interactions between neighboring monomers  (Fig.
1B). As a ¢rst step, two opposite recombinases catalyze single
strand breaks in the spacer region by phosphoryl transfer to a
tyrosine resulting in the formation of a free hydroxyl group.
The hydroxyl group attacks the intact and complementary
DNA strand on the other loxP site leading to the formation
of a Holliday junction. A similar mechanism resolves this
structure and leads to the formation of two new loxP sites.
This event is conservative; it does not cause any nucleotide
addition or removal, thus leaving intact the new loxP sites
that can be reused for further recombination events.
The conservative nature of the reaction makes it reversible.
0014-5793/02/$22.00 ? 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
PII: S0 014-57 93(02)032 66-0
*Corresponding author. Fax: (33)-1-44 27 13 22.
E-mail address: firstname.lastname@example.org (F. Tronche).
FEBS 26492FEBS Letters 529 (2002) 116^121
However, if the two loxP sites are located on the same DNA
molecule, their respective orientation determines the reaction
products resulting either in the inversion or the excision of the
intervening DNA segment (Fig. 1C).
The simplicity of this system makes it suitable for use in
organisms other than bacteria. Indeed, the pioneering work by
B. Sauer demonstrated that it e⁄ciently works in organisms
with very large genomes . Today it is widely used as a tool
for DNA rearrangement in plants, insects and mammals [7^
3. Conditional gene activation or inactivation in mice
In mice, the Cre-loxP system was initially used to switch on
gene expression in a given cell population [1,2]. Two distinct
transgenic mouse lines were generated. The ¢rst carries a si-
lent transgene, that is spaced from a promoter by a ‘stop
cassette’ (Fig. 2A). The stop cassette prevents transcription
of the transgene because it contains either a strong polyade-
nylation signal and/or a splice donor sequence or it disrupts
the ORF of the silent gene [1,2,11]. The second line carries a
transgene that drives the expression of Cre recombinase in a
cell type-speci¢c way. In every Cre expressing cell, the stop
cassette will be excised enabling the expression of the desired
transgene exclusively in those cells. This approach has many
applications. For example, one can generate transgenic lines
that will express toxic proteins, follow the destiny of cells
irreversibly marked by the expression of a reporter gene, or
determine the recombination pattern of a speci¢c Cre line
using L-galactosidase or £uorescent proteins as reporter. The
only limitations are those inherent to transgenesis. When
small transcriptional regulatory DNA regions are used, the
expression might be ectopic, mosaic and sometimes varie-
gated, due to the in£uence of genomic surroundings at the
integration site . In addition, transgenes from prokaryotic
origin may be poorly translated and subject to extinction in
mice . Recent developments, such as the characterization
of a locus that can be targeted in ES cells and allows ubiq-
uitous expression , the use of very large DNA segments
(YACS and BACs ) as vectors for transgenesis, and the
generation of ‘humanized’ ORF, overcome these problems
The possibility of cell-speci¢c inactivation of a desired gene
within the organism is a unique tool for addressing the func-
tion of genes involved in complex physiological networks and
for dissecting their respective roles in di¡erent tissues. Since its
¢rst description in 1994 by Gu et al. , a growing number
of studies have further developed this strategy  (Figs. 2B
and 3A). It consists of inserting into the target gene, by ho-
mologous recombination in ES cells, two loxP sites in the
same orientation and at a strategic position, thus ensuring
that removal of the intervening DNA segment results in com-
plete inactivation of the gene. The inactivation pro¢le depends
on the expression pro¢le of the recombinase (Fig. 3A). It is
essential that the insertion of the loxP sites does not interfere
with the normal expression of the gene. Ideally, they should
be placed in introns or non-transcribed regions, avoiding the
disruption of regulatory regions. However, in several cases a
loxP was inserted in transcribed but untranslated regions
without negative e¡ects. If the distance between the two
Fig. 1. Mechanism of site-speci¢c Cre recombination. A: The loxP site consists of two inverted 13 bp Cre binding sites that surround a central
8 bp spacer. A red arrow indicates the orientation of the loxP site. Cre recombinase is pictured in green. The ¢rst cleavage reaction is pictured.
B: Model of the Cre-loxP recombination pathway. Four Cre molecules form a synaptic tetramer. The tyrosines 324 from two of them cleave
the DNA backbone. The released 5P OH ends attack the partner strand to form a Holliday intermediate. A second round of cleavage and
strand exchange results in the recombinant products [5,67]. C: If the two recombination sites are in the same orientation the strand exchange
leads to excision or integration. If they are in the opposite orientation the outcome is an inversion.
F. Tronche et al./FEBS Letters 529 (2002) 116^121
loxP sites was a concern when designing the ¢rst conditional
alleles, it is now clear that, even if the e⁄ciency of site-speci¢c
recombination drops as a function of the distance, very large
DNA segments (up to 4 Mb) can be e⁄ciently excised by Cre
in vivo [18,19]. It is advisable to remove any DNA cassette
used during ES cells targeting since it may interfere with the
expression of the targeted or neighboring genes . This was
initially done using a targeting construct harboring three loxP
sites, two of which were £anking the selection cassette. Sub-
sequently, a transient expression of Cre provided ES cells in
which a partial recombination event took place, thus excising
the cassette but leaving the two loxP sites intact . New
vectors in which FRT sites £ank the selection cassette facili-
tate this step . The cassette can be removed by expression
of the £p recombinase in the ES cells or by mating animals
carrying the targeted allele with mice that express the £p in
the germ line  (Fig. 2E).
A precise determination of the recombination pro¢le is very
important for interpreting any phenotype. Unfortunately, the
expression pattern of Cre or the recombination pattern of a
reporter gene do not necessarily predict the recombination
pattern of the targeted gene because the e⁄ciency of Cre-
loxP recombination also depends on the genomic integration
sites of the loxP sequences . Ideally, the disappearance of
the gene product should be followed using antibodies (Fig.
3A). In some cases, depending on the structure of the gene,
an alternative strategy has been used, which couples the ex-
cision event with the activation of a silent reporter gene in-
serted at the targeted locus .
Finally, an important consideration is the time course of the
disappearance of the gene product that is a function of the
stability of mRNAs and proteins present in the cells at the
time of the gene deletion. If recombination takes place in
dividing cells, the disappearance of the gene product will be
facilitated by dilution due to cell division. In non-dividing
cells this can be a major problem especially in the case of
very stable mRNAs or proteins that are not actively degraded.
4. Chromosomal rearrangements
Cre recombinase proved to be useful for the design of chro-
mosomal aberrations such as deletions, inversions, transloca-
tions and duplications [19,25^27]. It can be used as a model
for human chromosomal disorders associated with chromo-
somal deletions and duplications or with translocations as
seen in some tumors. Chromosomal rearrangements can be
used to establish balanced lethal systems to facilitate stock
maintenance , large deletions can be used for genetic
screening of recessive mutations, and translocations can be
used to obtain mosaic animals carrying wild-type, heterozy-
gous and mutant cells after the induction of mitotic sister
chromatids exchange . For these purposes, two loxP sites
have to be introduced into the genome. If they are inserted in
the same chromosome, depending on their orientation, Cre
will mediate large deletions or inversions. If they are targeted
on two heterologous chromosomes, the presence of Cre will
lead to a balanced translocation (Fig. 2C). Finally, if they are
targeted on two homologous chromosomes but positioned at
di¡erent locations, the action of Cre will generate a deletion,
in one, and a duplication, in the other, homologous chromo-
some (Fig. 2D). The e¡ect of these rearrangements can be
tested directly in chimeric animals generated from the modi-
¢ed ES cells or eventually, when possible through germ line
transmission. In ES cells, the rare occurrence of rearrange-
ments makes a selection scheme based on the reconstitution
of a selection gene (usually hprt) necessary . However,
even without selection, some of these rearrangements can e⁄-
ciently be induced in animals when Cre is expressed from a
transgene and balanced translocations between heterologous
chromosomes have been observed at a low e⁄ciency . In
addition, translocation between homologous chromosomes
could be dramatically increased using an elegant experimental
design that takes advantage of chromosomal pairing during
meiosis and allowed the generation of balanced deletion and
duplication of large DNA segments  (Fig. 2D).
Fig. 2. Application of site-speci¢c recombination as a genetic tool in mammals. For A^H details are described in the text. LoxP sites are indi-
cated by red arrows and Cre recombinase in green. If the loxP site is located in the 5P untranslated region (A), an inverted orientation is advis-
able as it prevents the introduction of ATG codons in front of the expressed ORF . In E, instead of loxP sites also FRT sites could be
used. The star indicates as an example an introduced point mutation. The Cre-loxP system can also be used to resolve transgene silencing due
to concatemerization of transgene copies (F) . In G, the mutation of the loxP site in the Cre binding site is represented by a dot. In H, the
mutation in the loxP spacer is indicated by a black arrow.
F. Tronche et al./FEBS Letters 529 (2002) 116^121
5. Inducible somatic mutations in mice
Adding temporal control over the recombinase activity
would not only allow the generation of tissue-speci¢c muta-
tions that would be otherwise lethal, but also the study of the
physiology and behavior in the same animal before and after
the mutation. This avoids variations between individual or-
ganisms and limits the time for compensatory mechanisms.
Several strategies have been developed. One is to control the
recombinase activity. This requires the fusion of Cre ORF to
a mutated form of the ligand-binding domain (LBD) of a
nuclear receptor (progesterone (PR), estrogen (ER) or gluco-
corticoid (GR) receptors [32^34]). The mutated LBDs bind
synthetic steroid analogs (RU486, tamoxifen and dexametha-
sone, respectively) but do not bind endogenous steroids, thus
preventing the induction of recombination by endogenous
hormones. If Cre is fused to a PR-LBD, the activity of the
CrePR fusion protein is strongly reduced due to binding of
chaperones. Injection of RU486 to an adult animal releases
the fusion protein and results in Cre-mediated recombination
(Fig. 3B). When tested in cell culture, comparable CreER and
CrePR fusion proteins displayed similar induced and back-
ground activities . Both systems can regulate site-speci¢c
recombination in di¡erent organs, such as the blood, the
brain, the heart, the liver and the skin [32,33,35^39]. It is
di⁄cult to compare the two systems in vivo since the fusion
proteins, their expression levels, their targets and the cell types
were di¡erent. However, the tightest control was observed
with CreER expressed in the skin. Recently, for both systems,
new fusion proteins that are more tightly regulated and more
sensitive to the inducer have been developed ([33,40] and E.C.,
Another strategy is to control the expression of the recom-
binase using inducible promoters. The Mx1 promoter that can
be induced by application of interferon or poly dIdC to mice,
and tetracycline dependent arti¢cial promoters that have the
advantage to allow tissue speci¢city have both been used
[41,42]. Finally, to induce a mutation at a particular time,
the recombinase can also be introduced using viral transduc-
tion with vectors derived from viruses , or it can be fused
to membrane translocation sequences that allow a protein to
enter a cell simply by crossing the cell membrane .
6. Targeted insertions
Targeting of transgenes into a de¢ned genomic location
abolishes the variability of transgene expression due to posi-
tional e¡ects. Cre catalyzes both integration and excision but
the monomolecular nature of the excision favors the excision
reaction. Therefore, although integration events can be se-
lected in cell culture, their low occurrence does not allow
the use of this approach in vivo. Several attempts have been
performed on this system to reduce the e⁄ciency of the ex-
cision step and thus favor integration. Recombination be-
tween a pair of loxP sites, one carrying a mutation on the
left 13 bp repeat and the other one on the right repeat, gen-
erates an intact and a doubly mutated loxP site (, Fig.
2G). This reduces, but unfortunately does not abolish, the
reverse excision. An alternative approach would be the use
of an unidirectional recombinase that in contrast to Cre per-
forms only one reaction (the integration in this case). The
phiC31 integrase is such a recombinase and is active in mam-
malian cells . Another approach is the so-called ‘recombi-
nation-mediated exchange of cassette’ (RMCE) that has the
advantage of not integrating vector sequences. It relies on the
use of loxP sites mutated in the 8 bp spacer region. Cre cata-
lyzes recombination between two mutant sites but only poorly
between a mutant and an intact site. As picture in Fig. 2H, a
double recombination between two mutant and two wild-type
loxP sites in trans leads to the cassette exchange [47,48]. In
one study, RMCE has been demonstrated in the genome of
Fig. 3. Example of somatic mutagenesis of the glucocorticoid recep-
tor using site-speci¢c recombination in mice. A: Conditional inacti-
vation of the glucocorticoid receptor (GR) gene. The gene and
protein structures of this transcription factor are indicated. The
presence of loxP sites (red arrows) in the GRloxPallele makes it sen-
sitive to Cre-mediated inactivation. Cre activity deletes the third
exon including the DNA-binding domain. In addition, it introduces
a frameshift after the second and fourth exons are fused. The ex-
pression of Cre in the brain or in the liver speci¢cally depletes GR
protein levels in the corresponding tissues, as shown by immunohis-
tochemistry using an antibody directed against GR [69,70]. B: In-
ducible site-speci¢c recombination in the brain. See details in the
text. The CrePR fusion protein is expressed in the brain under the
control of the CamKIIK promoter. Injection of RU486 activates the
recombinase and results in the expression of a Cre dependent LacZ
F. Tronche et al./FEBS Letters 529 (2002) 116^121
injected mouse fertilized oocytes with a frequency of 15%.
However, since the mutation did not completely abolish the
recombination between heterospeci¢c loxP sites, excision sub-
sequently occurred . Further improvements, such as the
use of inverted loxP sites (the cassette is inverted but never
excised ) and more exclusive loxP mutations , are re-
quired to improve this approach in animals.
7. Some problems with the Cre-loxP system
Although the large number of publications using the Cre-
loxP system in mammals suggests that the recombinase is not
toxic, recent reports indicated that this assumption might not
be true. The attempt to express high levels of Cre in sperma-
tids resulted in sterility of the transgenic males, due to a very
high occurrence of chromosomal aberrations . Several oth-
er reports observed a decrease in cell proliferation as well as
an increase in apoptosis in cells expressing high levels of Cre
[43,53,54], which is associated with the accumulation of Cre
expressing cells in the G2/M phase of the cell cycle, chromo-
somal rearrangement and the appearance of micronuclei.
These aberrations could be due to the action of Cre on cryptic
target sites that exist in the genome . Alternatively, Cre
may induce nicks, which are then converted into double
strand breaks and repaired via non-homologous recombina-
tion resulting in the accumulation of fragmented DNA. To
prevent such deleterious e¡ects, special care has probably to
be taken on the level of Cre expression. Self-excising Cre
vectors may minimize the time window for the toxicity of
Cre, as long as it ensures recombination of the targeted locus
before disappearance of the recombinase [43,53].
Another concern is the fact that sensitivity of the loxP
target to recombination may vary from locus to locus .
In addition, a given target might be recombined at early
stages of development but not in adult tissues  or can
become insensitive to recombination, due to methylation
. Therefore, a given Cre line may recombine di¡erent tar-
gets with di¡erent kinetics and cellular speci¢cities. This can
intuitively be explained by di¡erential accessibility of the tar-
get site due to di¡erences in the chromatin state. These facts
illustrate that the recombination pattern obtained with one
conditional allele is not su⁄cient to predict the pro¢le of
recombination of another conditional allele, even if exactly
the same Cre line was used.
8. Other site-speci¢c recombinases
The parallel development of alternatives to the Cre/LoxP
system is necessary. The utilization of other recombinases
than Cre may allow studying sequential biological events by
introducing more than one somatic mutation into the same
animal (Fig. 4A). As depicted in Fig. 4B,C it also enhances
the potency of site-speci¢c recombination. In the ¢rst example
recombinase A inverts a DNA segment and this inversion is
stabilized by the presence of recombinase B, allowing one to
swap the expression of an intact exon with that of a mutated
one. In the second example, a cell type-speci¢c recombination
is achieved by combining the expression patterns of two di¡er-
ent gene promoters. Recombination would occur only in cells
that express both recombinases.
Two strategies have been followed to develop further re-
combinases that are active in mammalian cells. The ¢rst is
to take advantage of the diversity of existing recombinases
in nature, the second to modify the existing ones. The yeast
Flp recombinase and its target, FRT, do work in mice, in
particular the mutated version that was selected for higher
e⁄ciency at 37‡C  and has been widely used recently to
remove selection cassettes after homologous recombination in
mice . Recently, other recombinases from yeast  or
bacteriophages (R4, lambda, phi31, HK022 and TP901-1
[46,59,60]) have been also demonstrated to catalyze recombi-
nation events in mammalian cells. In parallel, new recombi-
nases were obtained by mutating the Cre, generating chimeric
proteins generated from Cre and £p enzymes or by accelerated
protein evolution [61^63]. In the latter case, the authors were
able to select for a Cre variant that recognizes a DNA target
naturally present in the human genome, opening up the pos-
sibility of speci¢cally targeting existing DNA sequences in the
genome for genetic modi¢cations.
The use of site-speci¢c recombinases allows us to examine
the consequences of sophisticated genetic modi¢cations at the
level of an entire mammalian organism. Conditional muta-
genesis is however tedious and requires long breeding
schemes. It will provide answers to speci¢c questions with
an exquisite precision, but the parallel development of other
approaches, including the targeting of gene products by small
molecules, RNA interference and systematic mouse mutagen-
esis [64^66], is essential to get an insight into genetic networks,
by targeting simultaneously several partners, at a genome
Acknowledgements: The FRE2401 is supported by the CNRS, the
MENRT, the FRM, and the AFM. I.S. and M.T. are fellows of the
‘Fondation Del Duca’ and the MILDT, respectively. The experiments
Fig. 4. Possible applications of using two distinct site-speci¢c recom-
binases. See the text for comments.
F. Tronche et al./FEBS Letters 529 (2002) 116^121
presented in Fig. 3 were performed in G. Schu «tz’s laboratory and we
wish to thank him for support. We apologize to all colleagues whose
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