Inducible Gene Expression and Gene Modification in Transgenic Mice
FRÉDÉRIC JAISSER
INSERM Unit 478, Medicine Faculty Xavier Bichat, Paris,
France.
Correspondence to Dr.
Frédéric
Jaisser, INSERM Unit 478, Medicine Faculty Xavier Bichat, 16 rue Henri
Huchard, 75018 Paris Cedex, France. Phone: 33-1-4485-6320; Fax:
33-1-4229-1644; E-mail:
jaisser{at}bichat.inserm.fr
Abstract. Animal transgenesis has proven to be useful for
physiologicas well as pathophysiologic studies. Animal models with
conditionalexpression of a transgene of interest or with a conditionalgene
mutation can be generated. This permits spatial and temporalcontrol of the
expression of the transgene or of gene mutationspreviously introduced by gene
targeting. These approaches allowthe generation of models suitable for
physiologic analysis ormodels mimicking disease states.
In vivo analysis of gene function is often based on the production
ofanimal models with specific genetic modifications. These approacheshave
been successfully applied to several areas in the renalfield
(1). Such modifications can
result in a gain or loss offunction. Two strategies are used to achieve these
goals (2).The first is
additive transgenesis, which consists of the introductioninto the genome of a
transgene, i.e., a fusion gene containinga promoter sequence and a
cDNA of interest. The promoter conferswidespread or tissue-specific
expression of the cDNA. The cDNAof interest can encode a wild-type protein or
a mutated proteinacting as a negative-dominant protein or mimicking genetic
mutationsobserved in human diseases. The second approach is gene targeting,
whichconsists of the introduction of specific mutations into a geneof
interest by homologous recombination using embryonic stemcell technology
(3,4).
In most cases, the mutation results inthe generation of a null allele
i.e., knockout, allowing geneinactivation after breeding of the
animals as homozygotes. Truncatedor mutated proteins can also be produced
using gene targeting.Finally, using a similar technique (i.e.,
knockin), the expressionof a protein of interest can be placed under the
control ofthe endogenous regulatory sequences of a different gene
(3,4).
In all of these cases, the expression of the wild-type or mutatedprotein,
as well as the site-directed gene modification, isconstitutive. The gene
mutation is present in the first cellof the embryo and thereafter in all
cells of the adult animal.Spatiotemporal restriction of the expression of the
proteinof interest is linked to the properties of the promoter usedto make
the fusion gene (in the case of additive transgenesis)or by the expression
pattern of the gene that has been mutated(in the case of the knockin
technique). Therefore, the proteinis expressed when the promoter used in the
transgenic constructis turned on, and its expression pattern follows the full
expressionpattern of the promoter used.
For more precise analysis of the function of a gene product,it may be
disadvantageous that the transgene or the mutationis expressed early during
development or in several organs,rather than one of interest. For example,
toxic effects or lethalityrelated to early expression of the transgene during
embryonicdevelopment may impair analysis of the phenotype. Moreover,as often
observed in knockout experiments, redundancy amongrelated gene products can
severely reduce the usefulness ofsuch animal models. To overcome these
limitations, several strategieshave been developed to allow spatiotemporal
control of the expressionof proteins or gene mutations. Among these
approaches, severalhave been proven to be functional in vivo in
transgenic animals,allowing investigators to predict and control where and
whentransgenes or gene mutations are expressed.
To date, two major systems have been successfully used in transgenicmice,
i.e., the tetracycline-inducible system and the Cre/loxPrecombinase
system (either constitutive or inducible). Otherinducible systems have been
described and used in transgenicmice
(5). Because of editorial
limitations, they are not detailedin this short review.
Both systems [tetracycline-controlled transactivator (tTA)/reverse tTA
(rtTA) and Cre/lox] can be used in cell culturemodels transfected
with the appropriate constructs. To use thesesystems in vivo, it is
necessary to generate two sets of transgenicanimals. One mouse line expresses
the activator (tTA, rtTA,or Cre recombinase) under the control of a selected
tissue-specificpromoter. Another set of transgenic animals express the
"acceptor"construct, in which the expression of the transgene of
interest(or the gene to be modified) is under the control of the target
sequencefor the tTA/rtTA transactivators (or is flanked by loxP sequences).
Matingthe two strains of mice allows spatiotemporal control of transgene
expressionor the desired gene alteration.
The tetracycline-dependent regulatory systems (tet systems)
developedin the laboratory of Herman Bujard permit stringent controlof gene
expression in a wide range of cells in culture, as wellas in transgenic
animals
(6,7,8,9).
The tet systems rely ontwo components, i.e., a
tetracycline-controlled transactivator(tTA or rtTA) and a tTA/rtTA-dependent
promoter that controlsexpression of a downstream cDNA, in a
tetracycline-dependentmanner (Figure
1A). tTA is a fusion protein containing the repressorof the Tn10
tetracycline-resistance operon of Escherichia coliand a
carboxyl-terminal portion of protein 16 of herpes simplexvirus (VP16). The
tTA-dependent promoter consists of a minimalRNA polymerase II promoter fused
to tet operator (tetO) sequences(an array of seven cognate
operator sequences). This fusionconverts the tet repressor into a
strong transcriptional activatorin eukaryotic cells.
Figure 1. Diagram of the tetracycline-inducible expression system. (A) The mechanisms
of action of the tet system are shown. The tetracycline-controlled
transactivator (tTA) is a fusion protein containing the tet repressor
of E. coli and the carboxyl-terminal domain of the VP16 transcription
factor from herpes simplex virus. tTA binds to an array of seven cognate
operator sequences (tetO) and activates transcription of a minimal
human cytomegalovirus promoter. In the presence of tetracycline or its
derivatives [such as doxycycline (Dox)], tTA cannot bind its target and the
system is inactive (tet-OFF system). Depending of the promoter used
in the tTA fusion gene, tTA is expressed ubiquitously or with a
specific-tissue expression pattern. (B) A mutated tTA, i.e., reverse
tTA (rtTA), has been obtained. In this case, the presence of doxycycline
allows the system to be turned on (tet-ON), in contrast to the
tet-OFF system. (C) In vivo, the system requires two
independent types of transgenic mice to be generated, i.e.,
transactivator mice, in which tTA or rtTA is expressed under the control of a
tissue-specific promoter (Psp), and responder mice, in which expression of the
cDNA of interest is under the control of the tetO minimal promoter.
Breeding generates double-transgenic mice. Addition or no addition of
doxycycline to the food or drinking water of the double-transgenic mice allows
in vivo spatiotemporal control of cDNA expression.
In the absence of tetracycline or its derivatives (such as doxycycline),
tTAbinds to the tetO sequences, allowing transcriptional activation
ofthe tTA-dependent promoter. However, in the presence of doxycycline,tTA
cannot interact with its target and transcription does notoccur. The
tet system that uses tTA is termed tet-OFF, because
tetracyclineor doxycycline allows transcriptional down-regulation
(6,7).
Becausetetracycline and its derivatives are not usually present inliving
animals, exogenous administration of tetracycline orits derivatives allows
temporal control of transgene expressionin vivo. A mutant form of
tTA, termed rtTA, has been isolatedusing random mutagenesis
(8,9).
In contrast to tTA, rtTA isnot functional in the absence of doxycycline but
requires thepresence of the ligand for transactivation. This tet
systemis therefore termed tet-ON
(Figure 1B).
The two systems function as mirror images and are functionallyequivalent
when transferred into mice (Figure
1C). It shouldbe noted that tet-ON requires higher
doxycycline concentrationsto be active, compared with the concentration
tet-OFF requiresto be inactive
(9). This may be of importance
when the systemsare used in vivo, because the doxycycline may differ
among tissues.The advantages of tet-ON, compared with
tet-OFF, are that thetransgene is not expressed until doxycycline is
given to theanimals and that upregulation in vivo is faster than
downregulation.Hallmarks of the tet systems are the tightness of
control, theability to regulate gene activity in a tissue-specific manner,
thedoxycycline dose-dependent responses, and the ability to returnto a
control situation by simply discontinuing doxycycline administration.However,
the major disadvantage of the tet systems is that controlof the
expression of the acceptor construct is often leaky,because of strong
positional effects on the tetO minimal promoter.This requires the
generation of several acceptor mouse strainsto identify those that express
the transgene not constitutivelybut in an inducible manner. Several recently
published reportsdescribed improvements of the tet systems
(transactivators withless toxicity or different ligand sensitivities,
bidirectionaltetO minimal promoters, and reduced leakiness)
(10,11,12,13,14).
The tet systems have been used in vivo for the inducible
expressionof several transgenes, encoding, for example, reporter genes,
oncogenes,or proteins involved in the signaling cascade. These modelsare
listed in Table 1. The tTA and
rtTA systems are both activein the kidney. In whole-kidney extracts, 1,000-
to 10,000-foldactivation of luciferase activity was observed
(6,9).
In thatcase, expression of the tTA and rtTA transactivators was underthe
control of a strong promoter (cytomegalovirus IE). We arecurrently analyzing,
in such mice, the cell-specific expressionof the tTA and rtTA
transactivators, as well as the inducibleexpression of a reporter gene along
the nephron (S. Puttini,A. Beggah, and F. Jaisser, manuscript in
preparation). Thiswill permit the specific use of these existing transgenic
micefor the inducible expression of transgenes of interest in renal
pathophysiologicmodels. To obtain more specific expression of the
tet systemsin the kidney, we are currently generating transgenic
mice inwhich expression of the rtTA transactivator is restricted tothe
collecting duct.
The Cre/lox system uses the Cre recombinase isolated from theP1
bacteriophage
(3,15).
The Cre recombinase catalyzes site-specificrecombination by crossover between
two distant Cre recognitionsequences, i.e., loxP sites
(Figure 2A). The loxP
sites includetwo 13-bp inverted repeats separated by an 8-bp spacer sequence.
AnyDNA sequence introduced between the two 34-bp loxP sequences
(termed"floxed" DNA) is excised because of Cre-mediated
recombination.Therefore, control of Cre expression in a transgenic animal,
usingeither spatial control (with a tissue- or cell-specific promoter)or
temporal control (with an inducible system), results in thespatial or
temporal control of DNA excision between the twoloxP sites. In
addition to conditional gene inactivation (conditionalknockout), this
approach can be applied to protein over-expression.In that case, a floxed
stop codon is inserted between the promotersequence and the cDNA of interest
(Figure 2B). Transgenic animals
donot express the transgene until Cre is expressed, leading toexcision of
the floxed stop codon. This strategy has been successfullyused to develop
reporter mice that express LacZ after Cre-mediatedrecombination
(16). This system has been
applied to tissue-specificoncogenesis
(17) and controlled antigene
receptor expressionin B lymphocytes
(18).
Figure 2. Diagram of the Cre/lox system and its use as an inducible
expression system. (A) The Cre recombinase has been isolated from the P1 phage
of E. coli. Cre induces recombination between two 34-bp,
site-specific recognition sequences (loxP), allowing excision of the
DNA flanked by these loxP sites (floxed DNA). In transgenic mice,
expression of Cre recombinase is under the control of ubiquitous or
tissue-specific promoters (P) or follows transient infection with a Cre
adenovirus. This allows conditional gene targeting when two loxP
sites have been introduced into the genome by homologous recombination. (B)
Conditional gene expression can be achieved using Cre recombinase. In this
case, a floxed stop sequence is placed between the promoter and the cDNA to be
expressed. In double-transgenic mice, the cDNA cannot be expressed in tissues
or cells in which Cre recombinase is not expressed. However, Cre recombinase
expression results in excision of the floxed stop sequence and allows the cDNA
to be expressed. (C) Temporal control of Cre recombinase activity can be added
to the spatial control described in B. Chimeric recombinases with the
ligand-binding domain of several steroid hormone receptors have been produced.
The activity of the chimeric fusion protein is dependent on ligand binding to
the ligand-binding domain. Mutated ligand-binding domains have been used to
avoid Cre recombinase activation by endogenous steroids rather than exogenous
ligands such as tamoxifen (a synthetic glucocorticoid agonist) or RU486 (a
synthetic antiprogesterone).
Inducible Cre recombinases have recently been developed
(15,19).
Arequirement for temporal control of Cre-mediated recombinationis that the
inducible Cre recombinase be activated not by endogenousligands but only by
an exogenous ligand that is introduced ata chosen time by researchers. The
inducible Cre recombinasesare fusion proteins containing the original Cre
recombinaseand a specific ligand-binding domain
(Figure 2C). The functional
activityof the Cre recombinase is dependent on an external ligand thatis
able to bind to this specific domain in the fusion protein.Binding of the
ligand is thought to produce conformational changesof the fusion protein
and/or changes in the intracellular localizationassociated with targeting of
the recombinase to the nucleus
(15,19).
Initially, fusion proteins containing the Cre recombinase andthe
hormone-binding domain of nuclear receptors, such as receptorsfor
glucocorticoids, estrogens, or progesterone, were generated.To avoid
activation of the engineered Cre recombinases by endogenousligands, such as
circulating glucocorticoids, estrogen, or progesterone,mutated hormone
ligand-binding domains have been used. In suchcases, synthetic ligands bind
the modified Cre recombinasesbut endogenous ligands cannot.
Metzger et al.
(20) focused their interest on
Cre recombinasefusion proteins with the estrogen ligand-binding domain. A
mutatedform, which was previously demonstrated to bind synthetic antagonists
(suchas tamoxifen or its derivative 4-hydroxy-tamoxifen) but notcirculating
estrogens, was used. The so-called Cre-ERT recombinasehas been demonstrated
to be functional in vivo, allowing widespreadtime-dependent
recombination when Cre-ERT expression is placedunder the control of a strong
promoter, such as the cytomegalovirusIE promoter
(21). Tissue-specific
recombination was recentlyobtained by placing the expression of another
inducible Crerecombinase under the control of tissue-specific promoters.
Vasioukhinet al.
(22) designed a Cre
recombinase similar to Cre-ERT, usinga mutant form of the mouse estrogen
receptor-binding domain(termed Cre-ERTM). Expression of this inducible Cre
recombinasewas placed under the control of a human keratin 14 promoter,
allowingrestriction of inducible Cre recombinase expression to keratinocytes
(22).In an elegant
experiment, those authors demonstrated that recombinationcould occur after a
4-d intraperitoneal administration of 1mg of tamoxifen and also, in an even
more restricted way, aftertopical application of tamoxifen directly to a
limited areaof the skin of transgenic mice. Using a similar Cre recombinase,
Danielianet al. (23)
were able to induce conditional DNA recombinationin developing mouse embryos.
After injection of tamoxifen intopregnant mothers, the authors observed DNA
recombination inorgans expressing the Cre recombinase. Despite adequate
recombinationevents (observed in embryos), abortion often occurs, probably
becauseof the toxic effects of tamoxifen
(23). Indeed, one major
limitationof using tamoxifen in vivo may be its toxicity. To
minimizethis problem, Indra et al.
(24) recently developed a
secondgeneration of inducible Cre recombinase (termed Cre-ERT2), whichis
approximately 10 times more sensitive to 4-hydroxy-tamoxifenin vivo
than is the original Cre-ERT. Another disadvantage ofthe system is that it
may be somewhat leaky, resulting in constitutiverather than inducible
activation. To obtain spatial and temporalrestriction of the recombination
process, the activity of theengineered Cre recombinase should be strictly
dependent on thepresence of the exogenous ligand. Zhang et al.
(25) reporteda modified Cre
recombinase that included two mutated murineestrogen-binding domains, which
were located at the amino andcarboxyl termini of Cre (termed MerCreMer).
Those authors demonstratedthat the control of the recombination process was
more stringent,compared with other inducible Cre recombinases
(25,26).
Various nuclear hormone-binding domains have been used to produceinducible
Cre recombinases. The progesterone-binding domainhas been used in a variant
of inducible Cre recombinase. Theprogesterone-binding domain of the human
progesterone receptorhas been mutated to one that binds the synthetic steroid
RU486(a well known glucocorticoid receptor antagonist) but not endogenous
progesteroneor glucocorticoids
(27). The generation of
transgenic mice thatexpress that inducible Cre recombinase specifically in
the brain,using either calmodulin-dependent kinase II or Thy-1 promotersto
direct expression of the recombinase, allows conditionalknockout of the
glucocorticoid receptor, in a space- and time-dependentmanner
(27).
Another way to control the functional activity of the Cre recombinaseis to
control its expression level. In that case, Cre-mediatedrecombination is
dependent on the spatiotemporal control ofCre recombinase expression. Two
strategies have been reportedto date, namely the use of an inducible promoter
that directsexpression of the Cre recombinase and the use of a
bi-génicinducible system in which expression
of the Cre recombinasecan be induced in a ligand-dependent manner. In the
first approach,the expression of the Cre recombinase is placed under the
controlof the Mx1 promoter, whose activity is inducible by interferon
or , as well as by pI-pC (an interferon inducer). Cre recombinationwas
observed in vivo after pI-pC administration
(28). However,although
deletion of the floxed gene was almost complete inliver and spleen, DNA
recombination reached only 20 to 70% inother tissues, reflecting either poor
bioavailability of theinducer into such organs or restricted tissue-dependent
activityof the Mx1 promoter
(28). In the second approach,
the expressionof the Cre recombinase is under the control of a minimal
promoterwhose activity is dependent on the presence of a specific
transcriptionfactor
(29,30,31).
The functional activity of this transactivatoris dependent on the presence of
an exogenous ligand, i.e., doxycycline,as described above. This
system appears to be very powerful,because it combines efficient spatial and
temporal control ofCre recombinase expression and does not rely on the use of
aninducer with deleterious side effects. However, it should benoted that
this approach requires the in vivo combination ofseveral independent
transgenes or mutated loci. They includethe transactivator construct, the
acceptor construct (Cre recombinasein this case), and the transgene or locus
to be modified (onein the case of an expression construct or two in the case
ofconditional gene inactivation). Such a strategy would be ofpractical use
only if the transactivator and the acceptor constructsare combined within the
same transgene. This was recently reportedby Utomo et al.
(29), who demonstrated the
feasibility of sucha strategy for the spatial and temporal control of
Cre-mediatedDNA recombination (Table
1).
In recent years, several strategies have been developed to gainmore
precise spatial and temporal control of gene expressionand/or gene
modification. These strategies rely on the use ofsite-specific recombinases
and/or inducible systems. The twomajor systems currently in use,
i.e., the Cre/lox and tet systems,have been
described in this short review. Several new approachesare under investigation
and are expected to refine these powerfultools in the near future. It should
be noted that these sophisticatedsystems are difficult to handle and to
develop. However, theyare very effective for the elucidation of specialized
gene functionsor the creation of specific animal models for fundamental
researchor study of the pathogenesis of human diseases.
Acknowledgments
I am grateful to Nicolette Farman and Charles Babinet for helpfulcomments
on the manuscript. The contributions of Ahmed T. Beggah,Stefania Puttini, and
Antoine Ouvrard-Pascaud are especiallyacknowledged. This work was supported
by INSERM and by grantsfrom the Ligue contre le Cancer and the Association
pour laRecherche contre le Cancer.
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Top
Abstract
Introduction
or 
, as well as by pI-pC (an interferon inducer). Cre recombination was
observed in vivo after pI-pC administration
(28). However, although
deletion of the floxed gene was almost complete in liver and spleen, DNA
recombination reached only 20 to 70% in other tissues, reflecting either poor
bioavailability of the inducer into such organs or restricted tissue-dependent
activity of the Mx1 promoter
(28). In the second approach,
the expression of the Cre recombinase is under the control of a minimal
promoter whose activity is dependent on the presence of a specific
transcription factor
(29,30,31).
The functional activity of this transactivator is dependent on the presence of
an exogenous ligand, i.e., doxycycline, as described above. This
system appears to be very powerful, because it combines efficient spatial and
temporal control of Cre recombinase expression and does not rely on the use of
an inducer with deleterious side effects. However, it should be noted that
this approach requires the in vivo combination of several independent
transgenes or mutated loci. They include the transactivator construct, the
acceptor construct (Cre recombinase in this case), and the transgene or locus
to be modified (one in the case of an expression construct or two in the case
of conditional gene inactivation). Such a strategy would be of practical use
only if the transactivator and the acceptor constructs are combined within the
same transgene. This was recently reported by Utomo et al.
(29), who demonstrated the
feasibility of such a strategy for the spatial and temporal control of
Cre-mediated DNA recombination (Table
1). 
