An Irreplaceable Tool for the Study of Mammalian Development and Biology
CHARLES BABINET
Developmental Biology Unit, Pasteur Institute, URA CNRS 1960, Paris,
France.
Correspondence to Dr. Charles Babinet, Institut Pasteur,
Unité de Biologie du
Développement, URA CNRS 1960, 25 rue du
Docteur Roux, 75724 Paris Cedex 15, France. Phone: 33-1-4568-8559; Fax:
33-1-4568-8634; E-mail:
chbabi{at}pasteur.fr
Abstract. Stable integration into the mouse genome of exogenous
geneticinformation, i.e., the creation of transgenic mice, has
becomea privileged way of analyzing gene function in normal developmentand
pathology. Both gene addition and gene replacement may beperformed. This has
allowed, in particular, the creation ofmice in which precise mutations are
introduced into a givengene. Furthermore, in recent years, strategies that
induce theexpression of a mutation in a given type of cell and/or at agiven
time in development have been developed. Thus, the transgenicmethodology
affords a unique and irreplaceable tool for thestudy of mammalian development
and biology and for the creationof animal models for human genetic
diseases.
The ability to introduce new genetic information into the germlineof
complex organisms, i.e., the creation of transgenic organisms,has
completely revolutionized the study of all aspects of theirbiologic
processes. This is particularly true in the case ofmice, which represent a
favored model for the study of mammaliandevelopment and physiologic
processes. Here, I briefly reviewsome aspects of the generation and use of
transgenic mice, withspecial emphasis on targeted mutagenesis, which results
in thecreation of transgenic animals bearing mutations deliberately
introducedin a particular gene or genomic region of interest. Many reviewsof
various aspects of transgenic technology have been published,and only a few
of them (among the most recent, whenever possible)are quoted here; readers
are referred to the first comprehensivereview
(1) and to a recent book and
references cited therein
(2).
Transgenesis by Addition of Cloned DNA Sequences: Pronuclear
Microinjection
One route for the creation of transgenic mice, which was designedin the
early 1980s, involves direct microinjection of clonedDNA into the pronuclei
of fertilized eggs. This procedure producesmice in which the transgene is
stably integrated into the mousegenome (generally as tandem head-to-tail
arrays of variablelength). This situation allows monitoring of transgene
expressionin all cell types and at any desired time in the developingembryo
and postnatal animal. Indeed, it was soon demonstratedthat a given transgene
may be expressed specifically in timeand space. However, a disadvantage is
that transgene integrationis apparently random; therefore, neighboring
sequences mightinterfere, both qualitatively and quantitatively, with proper
regulationof the transgene under study. Such an effect is referred toas a
"position effect"
(3) and might obscure analyses
of transgeneregulation. Interestingly, specific DNA sequences, termed locus
controlregions, have been discovered in the vicinity of some genesthat
render transgene expression copy number dependent and positionindependent
(4). Furthermore, particular
sequences in the locuscontrol regions were recently demonstrated to act as
boundariesbetween active and inactive chromatin
(5). Despite the position
effect,it has become possible, by creating sufficient numbers of transgenic
micecarrying different regions of a gene, to delineate the
cis-actingregulatory sequences implicated in correct temporal and
spatialgene expression.
Fusion Genes and Targeting of Gene Expression
The availability of characterized regulatory sequences responsiblefor
tissue-specific expression allowed the creation of transgenicmice in which
the expression of a given gene is deregulated.This is achieved by the use of
fusion genes as transgenes. Dependingon the promoter/regulatory elements used
and the coding sequencesfused to them, enhanced or ectopic expression of
either thenormal gene product or a modified gene product may be obtained.
Therefore,the transgenic approach permitted gain-of-function genetic
modifications,the effect of which could yield insights into the biologic
and/orpathophysiologic role of the gene product under study. Otheruses of
fusion transgenes include the use of a reporter genesuch as Escherichia
coli-galactosidase to define expressionpatterns
(6) and the use of a toxic
agent to ablate a particularpopulation of cells, to obtain insights into
their biologicfunction
(7).
Transgenes as Insertional Mutagens
When any exogenous DNA is introduced into the mouse germline,it may be
integrated into the genome in such a way that it perturbsthe expression of an
endogenous gene, causing a mutation, whichmay result in a phenotype when
transgene insertion is renderedhomozygous. Systematic intercrossing between
transgenic micethus becomes a way of screening for insertional mutations. The
mainadvantage of this approach is that the transgene can serve asa molecular
tag to recover the affected gene (for review, seereference
(8). Indeed, despite the fact
that transgene insertionfrequently entails genomic rearrangements at the site
of insertion,several studies have illustrated the attractiveness of this
approach,and several new genes of interest could be isolated in thisway
(see, for example, references
(9 and
10) and referencescited
therein).
Embryonic Stem Cells and Generation of Programmed Modifications of
the Mouse Genome
Knockout
Despite their widespread use in genetic studies of mouse biologic
processes,gene-addition transgenic mice have important limitations; neither
thecopy number nor the site of integration is controlled, resultingin
difficulty obtaining reliable specific expression of thetransgene.
Furthermore, with the exception of rare mutationsobtained via serendipitous
integration of a transgene into anendogenous gene, only gain-of-function
mutations can be obtainedin these transgenic mice. These limitations were
overcome inthe late 1980s with the combination of two methodologies, onethat
resulted in the generation of embryonic stem (ES) cellsand the other that
allowed investigators to perform and identifyhomologous recombination between
incoming DNA and the homologousendogenous genomic sequence. ES cells were
derived from preimplantationembryos and were shown to retain, in culture, the
remarkableability to colonize a host embryo, including its germline
(11,
12).The availability of ES
cells, which can be cultured in largenumbers, yields the possibility of
selecting various geneticmodifications in culture and obtaining corresponding
mice viathe generation of germline chimeras. In particular, methodsthat
permit the replacement of an endogenous allele by a disruptedallele via
homologous recombination in ES cells have been developed
(13,
14).This is generally
achieved by the use of targeting vectors containingsequences of the gene of
interest and a selection cassette insertedin an exon, interrupting the unit
of transcription (Figure 1A).
Afterintroduction of the vector into ES cells, selection is performedand the
resistant clones are screened for the presence of homologousrecombinants,
which contain the disrupted allele of the geneunder study. Mice carrying the
disrupted allele are subsequentlyobtained via the generation of germline
chimeras. This approachhas yielded enormous possibilities for the study of
gene function.Indeed, >1000 genes (see the Transgenic/ Targeted Mutation
Database;http://tbase.jax.org/) have been targeted in this way, and thestudy
of the phenotypes of the mutant embryos or mice thus createdhas clarified the
function of genes in the development and physiologicprocesses of mice.
Figure 1. Knockout and knockin strategies. (A) Knockout. The targeting vector
contains a selection cassette inserted into an exon and surrounded by
sequences homologous to the endogenous allele. After homologous recombination
between the targeting vector and the endogenous homologous sequences, a null
allele is created. (B) Knockin. The method is as depicted in A, except that a
cDNA of interest is inserted in-frame in an exon of the gene to be targeted.
In the mutated allele, the gene of interest is expressed in place of the
endogenous gene product.
Knockin
An interesting extension of the knockout approach is the so-calledknockin
approach, in which the targeting vector contains, inaddition to the selection
cassette, a cDNA of interest insertedin-frame in an exon of the gene under
study (Figure 1B). ThecDNA is
thus expressed in place of the endogenous gene product.The main advantage of
this approach is that it allows tightcontrol of the expression of the cDNA of
interest, because thecDNA is placed in the context of the complete set of
cis-actingregulatory elements that normally control the expression
ofthe endogenous genes; furthermore, this approach avoids theposition effect
encountered in gene-addition transgenesis (seeabove). A common use of this
approach is the targeting of theLacZ reporter gene, which permits
accurate definition of theexpression pattern of endogenous genes (see, for
example, references(15 and
16). This approach has also
proven to be a powerfultool for studying the functional relationships among
membersof a gene family and their potential abilities to functionally
compensatefor each other (see, for example, reference
(17).
Other Types of Mutations
In the strategies described above, the modification of the targetlocus
governed by the design of targeting vectors is mediatedby the endogenous
recombination machinery of the ES cells. Inrecent years, an important
breakthrough has been made by takingadvantage of site-specific recombinases,
which were discoveredin lower organisms but were demonstrated to work
efficientlyin mammalian cells (for review, see reference
(18). Becauseof space
limitations, I focus only on the most widely used,i.e., the Cre
recombinase of P1 bacteriophage. This enzyme promotesrecombination via
recognition of a 34-bp, asymmetric, nucleotidesequence (termed loxP)
(Figure 2A). Depending on the
relativeorientation of the loxP sites, Cre may catalyze excision
(sameorientation) or inversion (opposite orientation) of the DNAsegments
lying between these sites (Figure 2, B and
C).
Figure 2. Cre/loxP system of recombination. (A) The loxP sequences
recognized by Cre are shown. The small arrows indicate the site of cleavage by
Cre in the loxP sequence, which initiates the process of
recombination. (B and C) The DNA segment is either deleted (B, same
orientation of the loxP sites) or inverted (C, opposite orientation
of the loxP sites).
The use of the Cre/loxP system has considerably widened thescope
of targeted mutagenesis in ES cells. First, it has beeninstrumental in the
creation of new types of mutations, in additionto null mutations.
"Clean" (i.e., devoid of a selection cassettethat might
interfere with gene regulation) and subtle (e.g.,point mutations)
mutations can be created. To this end, a targetingvector that bears the
desired mutation in the gene of interestand the selectable marker in an
intron, flanked by two loxPsites in the same orientation
("floxed" sequence), is constructed.ES cells targeted in the
endogenous gene are then identifiedand transfected with a Cre-expressing
vector, which promotesthe excision of the selectable marker; this results in
ES cellsbearing the desired mutation in the gene of interest, with the
presenceof only one loxP site as a foreign sequence
(Figure 3, C and D)(19) (for review, see
references 20 and
21). Alternatively,the floxed
selection cassette may be excised in vivo by crossingmale chimeras
bearing the targeted allele with mice expressingCre during early development
(Figure 4, lower)
(22,23);
it shouldbe noted that there exists an alternative method to create clean
mutationsby homologous recombination in ES cells, which relies on a
hypoxanthinephosphoribosyl transferase-based selection system
(Figure 3, A and B)(24) (for review, see
references 20 and
21). The abilityto create
subtle mutations allows refinement of the analysisof gene function;
furthermore, it allows the creation of murinemodels of human genetic
diseases, which are frequently attributableto subtle genetic modifications
(e.g., point mutations).
Figure 3. Generation of clean mutations. (A and B) Double replacement.
hprt- embryonic stem cells are used. In the first step
(A), a targeting vector, containing an hprt expression cassette, is
used. hprt+ clones [hypoxanthine/aminopterin/thymidine
(HAT)-resistant] are selected and screened for the presence of homologous
recombinants. In the second step (B), the targeting vector is composed of
sequences homologous to the endogenous gene of interest with a point mutation
(*). After homologous recombination, the hprt selection
cassette is removed, and the corresponding hprt- clones
(with the mutation) can thus be selected using 6-thioguanine. Note that the
second targeting event may be repeated with different types of mutations. (C
and D) Use of the Cre/LoxP system. The targeting vector contains a floxed
selection cassette and a point mutation (*). The ES clones
containing the corresponding modified allele are then transfected with a Cre
expressing vector that permits the excision of the selection cassette.
Figure 4. Use of the Cre/loxP system in vivo. (Upper) Conditional
knockout. In the A x B mice, Cre is specifically expressed in the
central nervous system (NCS) (using a Cre fusion gene with the appropriate
regulatory sequences); consequently, the floxed allele is deleted in the brain
but not in the other tissues of the animal. (Lower) Deletion of a floxed
sequence in all cells of the A x C mice. This approach is used, for
example, to remove a selection cassette
(Figure 3) and entails the
expression of Cre in female germline fusion genes, e.g.,
phosphoglycerate kinase/Cre or Zona Pellucida protein 3/Cre.
Another important use of the Cre/loxP system is the creationof
deletions, which are valuable tools for functional analysisof the mammalian
genome (for review, see reference
(25). First,two loxP
sites are introduced in the chosen loci, in the suitableorientation, via two
steps of homologous recombination; subsequentexcision of the chromosomal DNA
between the two loxP sites isinduced by transfection of a
Cre-expressing construct in EScells. An elegant in vivo extension of
this approach was recentlydescribed. Transgenic mice carrying an
Sycp-1 (encoding synaptonemalcomplex protein-1)/Cre fusion gene were
shown to express Crespecifically during the prophase of the male first
meiotic division(26);
crossing such transgenic mice with mice carrying loxPsites on
homologous chromosomes yielded double-transgenic maleanimals, which produced
sperm with deletions or duplicationsof the genomic region between the two
loxP sites. Progeny ofthese male animals were demonstrated to carry
these chromosomalrearrangements. This approach should provide a powerful
meansfor functional analysis of complex genomic regions
(27).
Cre/loxP System and Conditional Mutagenesis
Finally, and most importantly, the Cre/loxP system has provided
thepossibility of conditional mutagenesis, i.e., the ability to
inducedisruption (or expression) of any gene in any cell type and/orat any
time of development (for review, see references
(20 and
21).This is important for at
least two reasons. First, when a mutationentails embryonic lethality, the
study of gene function laterin development is precluded. Second, when a gene
exhibits acomplex pattern of expression, the study of its function maybe
obscured when all cells of an individual express the mutation,as is the case
in knockout mice; therefore, it may be valuableto induce the mutation in one
cell type or tissue at a time.The approach to achieve inducible gene
targeting is basicallysimple and involves two types of transgenic mouse
lines; thefirst bears the target gene (or gene segment) flanked by two
loxPsites in the same orientation and positioned in such a way that
itdoes not prevent normal gene activity (floxed gene); the secondline
contains a fusion transgene expressing Cre recombinase.When these two mouse
lines are crossed, depending on the promoter/regulatorysequences present in
the fusion transgene, the floxed gene isdeleted and a null mutation is
created in particular cells ortissues
(Figure 4, upper). It is
important to note that tworequirements must be fulfilled for conditional gene
targetingwith the Cre/loxP system, i.e., (1) the
floxed allele must becreated in such a way that it is still functional and
(2) targetingof Cre expression must be tightly controlled. One
valuable wayto address the latter issue could be to knockin the Cre coding
sequencesin genes whose specificity of expression is well known and
appropriatefor the particular question being addressed, as illustratedin a
recent study (28). Conditional
mutagenesis can be refinedto induce a mutation not only in a given type of
cell but alsoat a given time in development. To that end, two approachesare
being developed. The first relies on the production of fusionproteins
containing Cre and the ligand-binding domain of a steroidreceptor; in such
chimeric proteins, the activity of Cre becomeshormone dependent
(Figure 5). Therefore,
recombination betweenloxP sites could be controlled in cells
carrying the chimericCre by the addition of ligand. Such an approach was
demonstratedto be operational in cultured cells
(29,30)
and in animals;excision of a loxP-flanked segment in transgenic mice
expressingchimeric Cre with a mutated ligand-binding domain of the human
estrogenor progesterone receptor was demonstrated to be dependent onthe
administration of synthetic ligands (tamoxifen or RU486,respectively)
(31,32).
The second system was devised by Gossenet al.
(33). Those authors
demonstrated that a fusion proteincomposed of a mutated version of the
tetracycline repressorsequences linked to the herpes VP16 activating
sequences (reversetetracycline-controlled transactivator) could bind only in
thepresence of tetracycline to the tetracycline operator (tetO)DNA
sequences, thus promoting the activity of a LacZ or luciferase
reportergene. This result, which was originally observed in culturedcells
(33), was extended to
transgenic mice
(34,35).
It shouldbe possible to adapt this strategy to the control of Cre expression.
Therefore,transgenic mice that contain two transgenes, one expressingthe
reverse tetracycline-controlled transactivator and the otherconsisting of
tetO sequences and a minimal promoter linked tothe Cre gene, could
be generated. Depending on the specificityof the promoter used to express the
fusion protein, such transgenicmice should express Cre, after tetracycline
treatment, in agiven cell type at the time of treatment. Indeed, the
feasibilityof such a strategy was elegantly demonstrated in a recent study
(36).
Figure 5. Use of a Cre fusion protein (Cre-ind) to control the time of induced gene
targeting. (A) The agonist acts on the inactive ligand-binding domain/Cre
fusion protein and activates it. (B) In A x B double-transgenic mice
(containing the Cre/ind fusion gene and the floxed allele), the
floxed allele is deleted only after injection of the agonist.
Almost 20 years ago, the first transgenic mice were born fromzygotes
microinjected with cloned DNA. Many studies have sinceaddressed almost all
aspects of mouse biologic features usingthis transgenic approach, thus
illustrating its unique versatility.However, the development of homologous
recombination in ES cellshas considerably widened the scope of transgenic
technology.Indeed, the repertoire of genetic modifications that can be
introducedvia ES cells in the germline of mice is almost unlimited. Notonly
null mutations but also discrete and clean mutations (suchas point mutations
or micro-deletions or -insertions) as wellas chromosomal rearrangements,
including large deletions, inversions,or translocations, may be created.
Lastly, strategies have beendeveloped that allow the induction of mutations
restricted toa given cell type or tissue, permitting conditional mutagenesis.
Takentogether, the variety of tools designed to modify the mousegenome have
provided unprecedented opportunities for the studyof gene function in the
development and physiologic and pathophysiologicprocesses of this complex
organism. There is little doubt thatthese tools will continue to be refined
and will thus increasinglycontribute to our knowledge of mammalian biologic
processes.
Acknowledgments
I am very grateful to Patricia Baldacci for critical readingof the
manuscript.
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Top
Abstract
Introduction
-galactosidase to define expression patterns
(6) and the use of a toxic
agent to ablate a particular population of cells, to obtain insights into
their biologic function
(7). 
