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GREAT EXPERIMENTS
Gene targeting: Altering the genome in mice
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Mario Capecchi
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Gene targeting provides the means to create strains of mice with specific mutations
in virtually any gene. This methodology permits evaluation of the functions of genes
in an intact mammal and systematic genetic dissection of the most complex biological
processes, such as development, learning, and immunity. The primary problem in developing
gene targeting was how to generate a specific mutation in a mammalian cell without
altering any other site in the genome. This became possible when we discovered that
cultured mammalian cells are capable of homologous recombination, allowing a mutant
gene injected into a cell to replace the chromosomal copy. Parallel advances in
the isolation of embryonic stem cells made it possible to apply this technique to
cells with sufficient developmental potential to allow mutations to be introduced
into the mouse germline and their effects ultimately observed in whole animals.
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Background
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Our contributions to gene targeting proceeded in three phases, the first beginning
in 1977. At that time I was attempting to improve the efficiency with which new
genes could be introduced into mammalian cells. Wigler and Axel had just reported
the first successful transformation of mammalian cells with an exogenous gene, having
succeeded in transferring a herpes simplex virus thymidine kinase gene (HSV-tk)
into tk – cultured cells by calcium phosphate coprecipitation (1985).
Although an important contribution to somatic cell genetics, this procedure was
not efficient. Approximately one in one million cells exposed to the calcium phosphate-DNA
coprecipitate acquired the exogenous gene in a functional form. Using the same experimental
paradigm, I asked whether I could introduce tk genes into tk –
mouse fibroblasts by injecting DNA directly into their nuclei (1986). This
procedure proved extremely efficient. One third of the cells that received the DNA
stably integrated the functional tk gene at random positions within their
genomes and passed it on to their daughter cells. This high efficiency of DNA transfer
by microinjection made it practical for investigators to generate transgenic mice
containing random insertions of exogenous DNA in their genomes. This was accomplished
by injecting the desired DNA into nuclei of one-cell zygotes and allowing these
embryos to come to term in foster mothers (see Transgenic mice: Expression of foreign
genes in animals).
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Efficient functional transfer of the tk gene into cells required that it
be linked to other short viral DNA sequences. When considering how to improve the
efficiency of transformation of mammalian cells, I had thought it plausible that
viral genomes might contain bits of DNA that enhanced their ability to establish
themselves within a mammalian genome. I succeeded in finding such a sequence in
the genome of SV40, a simian DNA virus. When coupled to the HSV-tk gene,
the SV40 sequence increased the efficiency of conferring a tk+ phenotype
to tk – recipient cells by a factor of more than 100 (1986).
The enhancement did not appear to be the result of HSV-tk plasmid replication
within the host cell before integration, and the SV40 DNA sequences were integrated
into the host genome along with the HSV-tk gene. I concluded that the efficiency-enhancing
sequence from SV40 was either increasing the frequency with which the exogenous
DNA was integrated into the host genome, or increasing the probability that the
tk gene, once integrated, was being expressed. The SV40 sequence proved
to be what would later come to be called an enhancer, and these experiments contributed
to their definition (1987). Our early experience with them would prove
invaluable in developing a reliable targeting approach.
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Figure 1
A plasmid injected into a cell becomes integrated into its genome as a concatemer.
Two mechanisms are possible for concatemer formation.
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Although the ability to transform cells efficiently was itself extremely useful,
the observation that I found most fascinating from these early DNA microinjection
experiments was that when multiple copies of the tk plasmid were injected
into cells, they were always integrated into the genome as head to tail concatemers.
This structure resulted even though integration could apparently take place throughout
the host chromosomes. I realized that concatemers could be generated either by replication
(e.g., a rolling circle mechanism) or by homologous recombination among the injected
plasmids, as shown in Figure 1. We were able to prove that they were formed by homologous
recombination (1988).
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This was a startling discovery at the time, because it had always been assumed that
homologous recombination was restricted to germ cells, where its purpose was to
shuffle the parental genetic traits to ensure their broad dissemination among the
offspring. Finding evidence of homologous recombination in mouse fibroblasts implied
that somatic cells also possessed the necessary machinery. We suspected that the
homologous recombination machinery in somatic cells was very efficient, because
when we injected more than one hundred tk plasmid molecules per cell they
were all incorporated into a single, ordered head-to-tail concatemer. It was immediately
clear that if we could harness this machinery to carry out homologous recombination
between a newly introduced DNA molecule of our choice and the same DNA sequence
in a recipient cell's chromosome, we would have the ability to specifically mutate
or modify virtually any cellular gene.
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The second phase of our quest for gene targeting required that we become familiar
with the substrate preferences and reaction products of the cellular recombination
machinery. We did this by studying recombination between cointroduced DNA substrates.
These experiments showed that the endogenous recombination machinery could mediate
a wide spectrum of reactions. Although a distinct bias towards nonreciprocal reactions
was observed, both reciprocal exchanges and nonreciprocal recombination events were
apparent among the products of recombination (1989) (for a discussion of
recombination see Genes 2000 Breakage and reunion involves heteroduplex DNA
. Either type of reaction would suit our purpose. We also found that the ability
to carry out homologous recombination depends on a cell's position in the cell cycle,
showing a peak of activity in early S-phase (1990), and that linear DNA
molecules appeared to be better substrates for homologous recombination than circular
or supercoiled molecules (1991). Together, these studies demonstrated that
the cellular enzymatic machinery for recombination not only existed, but that when
optimized it was sufficiently efficient that it might indeed be exploited to mediate
homologous recombination between exogenous DNA sequences and chromosomal sequences.
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Figure 2
Regeneration of a functional neor gene by gene targeting. The
targeting vector and the chromosome contain inactivating mutations at different
locations within the neor gene. Homologous recombination between
the two mutant genes generates a functional neor gene within
the chromosome in about 1 in 1000 cells.
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The third phase of our effort was to devise a way to extend homologous recombination
to sites in the genome itself. To makethings as simple as possible in the initial
stages, we began with studies involving homologous recombination with specific target
sites that we had created within the chromosomes. It was clear prior to the initiation
of these experiments that the frequency of targeting to a single copy gene in mammalian
cells was likely to be low and that insertion of the targeting vector at sites other
than the target locus would be far more common (simply because there were so many
more of them). To permit detection of rare homologous recombination events, we designed
lines of recipient cells with specific target loci that would provide a selection
protocol to eliminate cells not containing the desired recombination products, as
outlined in Figure 2. The first step of this scheme required generation of cell
lines containing random insertions of a defective neomycin resistance (neor)
gene containing either a deletion or a point mutation. The second step involved
introducing into those cell lines a targeting vector carrying a neor
gene with an inactivating mutation different from that of the target neor
gene. Homologous recombination between the targeting vector and the cognate sequence
in the recipient cell genome would generate a functional neor
gene from the two defective parts. Cells containing this recombination product would
be resistant to the drug G418, which kills cells that lack a functional neor
gene.
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We generated recipient cell lines containing single copies of the defective neor
gene, lines containing multiple copies of the gene in head-to-tail concatemers and,
by inhibiting concatemer formation, lines with multiple defective neor
target genes, each located on a separate chromosome. The different cell lines allowed
us to evaluate how the number and location of targets within the recipient cell's
genome influenced the targeting frequency.
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Acquisition of neomycin resistance by cells injected with a neor
targeting vector occurred at a frequency higher than we anticipated (1991).
One in a thousand injected cells yielded progeny with functional neor
genes. Southern transfer and sequence analysis showed that the neomycin- resistant
phenotype was indeed due to correction of the defective chromosomal neor
gene by homologous recombination with the incoming DNA, as we had intended. Correction
of a point mutation by gene targeting occurred at a frequency that was five orders
of magnitude higher than the spontaneous reversion frequency. Correction of a deletion
mutation was effectively at an infinitely higher frequency relative to its spontaneous
reversion frequency, since this mutation was never observed to revert.
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The frequency with which the defective chromosomal neor genes
were corrected was shown to be independent of the number of neor
targeting vector molecules introduced into the recipient cells and also independent
of the number of target neor genes present within the recipient
cells. Thus, introduction of one molecule per cell (based upon an assumed Poisson
distribution of input DNA) yielded the same successful targeting frequency as injection
of hundreds of molecules per cell. These results suggested that the targeting frequency
might be limited by the availability of the machinery needed to mediate homologous
recombination rather than by the number of incoming vector molecules or the number
of targets within the recipient cell lines.
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We were encouraged by the observation that the targeting frequencies in independent
cell lines with target sites at various genomic locations were identical, suggesting
that a large fraction of the genome should be accessible to gene targeting. Having
established that gene targeting could be achieved in cultured mammalian cells and
having determined some of the parameters that influenced its frequency, we were
now ready to extend gene targeting to endogenous loci and to the whole mouse.
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The experiment
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At this point, the constraints of what we were trying to accomplish made us face
the problem of what cells to use in order to continue our work. We had developed
the targeting procedure in cells chosen largely for purposes of convenience. They,
however, lacked the developmental plasticity that would be required to provide a
way of converting a targeted cell into a mouse bearing the mutation. The obvious
approach was to perform gene targeting directly in one-cell mouse embryos. This
would both produce a mutant mouse and guarantee introduction of the targeted mutation
into the mouse germline so that the mutation could be propagated. Unfortunately,
this approach was impractical because of the low frequency of targeted homologous
recombination compared to random integration of the targeting vector. Instead, it
appeared that our only choice was to do gene targeting with some kind of cultured
cells so that we could select the relatively rare targeted recombinants. This approach
would require that we use cells with the greatest possible developmental potential,
since we would have to generate a mutant mouse by introducing targeted cells into
an early embryo, where they would have to be able to contribute daughters to all
of the tissues of the developing mouse, including the germline. Even with mutant
cells in all of its tissues, however, most of the cells in the mouse would lack
the mutation, so it would have to be bred in order to finally produce mice with
the mutation in every cell.
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It initially appeared that the only cells capable of meeting our requirements for
developmental plasticity were cultured multipotent embryonal carcinoma (EC) cells,
a cell line derived from tumors formed by early embryos. In the late 1970s, EC cells
appeared to offer the only potential avenue between the culture dish and the whole
animal. When these cells are introduced into a mouse preimplantation embryo, and
the embryo allowed to implant into the uterus of a foster mother, they contribute
to the formation of most somatic tissues. However, after attending seminars and
conferences on EC cell biology over a number of years, I became painfully aware
of the frustrations associated with attempts to obtain EC cell contributions to
the formation of the mouse germline. Finally, at a Gordon Conference in the summer
of 1984, I heard a discussion about ES cells (then called EK cells), cultured multipotent
embryonic stem cells capable of populating the mouse germline. These cells were
developed in Martin Evans's laboratory, and differed from EC cells in that they
were obtained from a normal early embryo rather than from a mouse tumor. In the
winter of 1985, I arranged to spend a week in Evans's laboratory to learn how to
culture ES cells and generate chimeric mice from them. It was just before Christmas,
a marvelous time to be in Cambridge, England.
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In the beginning of 1986, all of our effort switched to performing gene targeting
in ES cells. We also decided to use electroporation as the means of introducing
the targeting vectors into ES cells. Although microinjection was orders of magnitudemore
efficient than electroporation as means for generating cell lines with targeted
mutations, injection had to be done one cell at a time. With electroporation, 108
cells could be manipulated in a single experiment, and if successful, the methodology
of gene targeting would be more readily transferable to other investigators.
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Figure 3
The targeting vector contains a neor gene inserted into one
of the exons of the hprt gene. Following homologous pairing between the vector and
the hprt gene within the genome, a homologous recombination event replaces the genomic
sequence with vector sequences containing the neor gene. Because
the neor gene is within an exon, the hprt gene is inactivated.
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The first gene that we disrupted in ES cells was that for hypoxanthine phosphoribosyl
transferase (hprt), which we chose because we could select directly for cells containing
a disruption of it. Further, because the hprt gene is located on the X
chromosome and the host ES cell line was derived from a male mouse, only a single
locus had to be disrupted to yield hprt – cell lines. The strategy
that we employed is shown in Figure 3. We placed a neor gene
within an intron in the middle of cloned hprt genomic sequences, then introduced
the vector bearing the disrupted hprt gene into ES cells by electroporation,
and finally selected for cells resistant to both G418 and 6-thioguanine (6-TG),
a drug toxic to cells with a functional hprt gene. All recipient cell lines
selected in this way had lost hprt function as a result of targeted disruption of
the hprt locus (1992).
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Because we foresaw that the neor gene could potentially be used
as a positive selectable gene for the disruption of many genes, it was critical
that neor expression be mediated by an enhancer that would function
regardless of its location within the ES cell genome. It was here that our earlier
experience with what came to be known as enhancers proved of value. To encourage
such strong neor expression, the enhancer we used to drive it
was a duplicated, mutant polyoma-virus enhancer known to permit robust viral replication
in mouse embryonal carcinoma cells (1992). Subsequently, this strategy
of using a ubiquitously expressed, positive selectable marker has been used by many
investigators to disrupt a wide spectrum of genes in ES cells.
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Our experiments with the hprt gene showed that ES cells were able to mediate
homologous recombination. They also clearly demonstrated the need for the ability
to select targeted cells. Under optimal conditions, the absolute targeting frequency
of the hprt disruption was one cell carrying a targeted mutation per 105
cells that were electroporated. For each successfully targeted cell, approximately
one thousand contained a random insertion of the neor target
vector into the genome. The selection protocols worked, however, and, just as importantly,
did not alter the pluripotent state of ES cells in culture (as we demonstrated in
separate experiments). I believe these disruption experiments with the hprt
locus played a pivotal role in the development of the field by making other investigators
realize that specific modification of a mammalian genome in vivo was actually
possible. Many people were encouraged to explore the use of targeted gene disruption
as a means for determining the function of mammalian genes they were studying.
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The high ratio (~1-1000) of nonhomologous to homologous recombination events that
we had observed in the hprt disruption experiment presented a potential
problem for the general applicability of gene targeting. Because the disruption
of most genes is not expected to produce a phenotype, like hprt –
, that is selectable at the cell level, an investigator seeking a specific gene
disruption would therefore either need to conduct a tedious DNA screen through many
colonies of cells to identify the rare colony containing the desired targeting event,
or devise a selection scheme able to enrich for cells containing the targeting event.
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Figure 4
The targeting vector has a neor gene inserted into an exon of
gene X and an HSV-tk gene at one end. Homologous recombination inserts
the neor gene into the genomic copy of gene X, but the HSV-tk
gene is lost. Random integration inserts both the HSV-tk and the neor
genes into the genome. The HSV-tk gene will convert the drug FIAU into
a toxic compound, killing the cell.
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Late in 1986, I conceived of a very general strategy to enrich for cells in which
the homologous targeting event had occurred. The strategy, known as positive-negative
selection, uses two components (see Figure 4). One component is a positive selectable
gene, neor, used as a marker to select for recipient cells that
have incorporated the targeting vector anywhere in their genome (i.e., at the target
site by homologous recombination or at other sites by nonhomologous recombination).
The second component is a negative selectable gene, HSV-tk, which is located
at the end of the linearized targeting vector and is used to select against cells
containing random insertions of the targeting vector. Thus the "positive" selection
enriches for all recipient cells that have incorporated the introduced vector, and
the "negative" selection eliminates those that have incorporated it at nonhomologous
sites. The net effect is to enrich for cells in which the desired homologous targeting
event has occurred. The scheme was based on the critical observation, in earlier
experiments, that insertion of exogenous DNA at nonhomologous sites included the
ends of the targeting vector. Conversely, homologous recombination eliminates the
ends of the targeting vector, thereby discarding the "negative" selectable gene.
The strength of this enrichment procedure is that it is independent of the function
of the gene that is being disrupted and succeeds whether or not the gene is expressed
in the cultured recipient ES cell. We tested this scheme and proved it to be workable
in an experiment involving the disruption of the proto-oncogene int-2 (1993),
and positive-negative selection has since become the most frequently used procedure
to enrich for cells containing targeting events.
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The legacy
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The use of gene targeting to evaluate the functions of genes in the living mouse
is now a routine procedure and is used in hundreds of laboratories all over the
world. It is very gratifying to be able to pick up almost any major journal in the
biological sciences and find the description of yet another so-called knockout mouse.
The in vivo functions of well over 7,000 genes have been analyzed using
the gene targeting technology. This number is particularly impressive considering
that no concerted program was involved to generate this collection of mouse lines
with targeted mutations. Rather, it was accomplished as a cottage industry, by the
collective work of many individual laboratories.
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Figure 5
After introduction of the targeting vector into ES cells by electroporation, selection
for cells with a neor gene and against those with an HSV-tk
gene kills all cells but those with the successfully targeted gene. The surviving
cells are grown into a colony in order to generate enough cells to use in making
mutant mice (see Figure 6).
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Figure 6
Injected cells mix with cells that lack the mutation, resulting in mice with both
mutant and normal cells ("chimeric" mice). Dark brown coat color distinguishes those
descended from targeted cells. Note that the coat color of the foster mother makes
no difference since she does not share genes with her progeny. Uniformly brown progeny
of the chimeric mice are heterozygous for the mutation, and must be interbred to
produce homozygous mice.
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The gene targeting protocol is now generally performed as follows: First, the desired
DNA sequence alteration is introduced into a cloned copy of the gene of interest
by standard recombinant DNA technology. Second, as shown in Figure 5, the alteration
is then transferred, by means of homologous recombination, to the cognate genomic
locus in pluripotent, mouse embryo-derived stem (ES) cells and cultured recipient
ES cell lines carrying the alteration are selected. Third, ES cells containing the
altered genetic locus are injected into mouse blastocysts, which are in turn brought
to term in foster mothers, generating chimeric mice that are capable of transmitting
the modified genetic locus to their offspring. This generates mice heterozygous
for the mutation in every cell. Finally, if the intent is to evaluate the consequences
of any type of recessive mutation, heterozygous siblings are interbred to yield
mice homozygous for the mutation. Figure 6 outlines these steps, from the injection
of cultured ES cell lines containing the desired targeted gene modification to the
generation of germline chimeras and their offspring.
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Gene targeting in mice has thus far been used primarily to completely disrupt specific
genes in order to try to determine their function by creating the severest possible
phenotype. However, it can be used to manipulate the mouse genome in almost any
desired manner. For example, it is possible to generate an allelic series of mutations
in a specific gene to evaluate the effects of gain-of-function or partial loss-of-function
mutations, as well as the effect of complete loss-of-function mutations. To permit
the evaluation of multiple potential roles of a gene, particularly if the loss-of-function
allele compromises the embryo at early stages of development, the Cre-loxP and Flp/FRT
site-specific recombination systems, in concert with gene targeting, can be used
to restrict the effect of a mutation to specific cells, tissues, or temporal periods
(1994; 1995; 1996). There is no question that the mouse
is a very complex organism, but it provides a model very similar to ourselves, and
the broad range of genetic manipulations in it made possible through gene targeting
promises the potential for deciphering even the most elaborate of its biological
processes.
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The author
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Mario R. Capecchi is Distinguished Professor of Human Genetics and Investigator
of the Howard Hughes Medical Institute at the University of Utah School of Medicine.
Dr. Capecchi is a member of the National Academy of Sciences. His honors include
the Bristol-Myers Squibb Award for Distinguished Achievement in Neuroscience Research,
the Gairdner Foundation International Award for Outstanding Achievements in the
Field of Medical Science, the General Motors Corporation's Alfred P. Sloan Jr. Prize
for Outstanding Basic Science Contributions to Cancer Research, the Bio-Analytica
Prize for Distinguished Contributions to the Field of Molecular Biology (Germany),
the Kyoto Prize for Eminent Achievements in the Field of Life Sciences, the Franklin
Medal for Advancing Our Knowledge of the Physical Sciences, the Rosenblatt Prize
for Excellence in Academic Achievements, and the Baxter Award for Distinguished
Research in the Biomedical Sciences.
Mario Capecchi
Howard Hughes Medical Institute
University of Utah School of Medicine
15 North 2030 East Room 5100
Salt Lake City, UT 84112-5331
Phone: 801 581 7096
Fax: 801 585 3425
E-mail: mario.capecchi@genetics.utah.edu
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Last Revised on September 16, 2004
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- 1985 Wigler, M., Silverstein, S., Lee, L., Pellicer,
A., Chen, Y. and Axel, R. (1977). Transfer of purified Herpes Virus thymidine
kinase gene to cultured mouse cells. Cell 11, 223-232. PubMed Journal
- 1986 Capecchi, M. R. (1980). High efficiency
transformation by direct microinjection of DNA into cultured mammalian cells. Cell 22,
479-488. PubMed Journal
- 1987 Levinson, B., Khoury, G., Woude, G. V., and
Gruss, P. (1982). Activation of SV40 genome by 72-base pair tandem repeats
of Moloney sarcoma virus. Nature 295, 568-572. PubMed
- 1988 Folger, K. R., Wong, E. A., Wahl, G., and
Capecchi, M. R. (1982). Patterns of integration of DNA microinjected into
cultured mammalian cells: evidence for homologous recombination between injected
plasmid DNA molecules. Mol. Cell Biol. 2, 1372-1387. PubMed
- 1989 Folger, K. R., Thomas, K., and Capecchi,
M. R. (1985). Nonreciprocal exchanges of information between DNA duplexes
coinjected into mammalian cell nuclei. Mol. Cell Biol. 5, 59-69. PubMed
- 1990 Wong, E. A., and Capecchi, M. R. (1987).
Homologous recombination between coinjected DNA sequences peaks in early to
mid-S phase. Mol. Cell Biol. 7, 2294-2295. PubMed
- 1991 Thomas, K. R., Folger, K. R., and Capecchi,
M. R. (1986). High frequency targeting of genes to specific sites in the mammalian
genome. Cell 44, 419-428. PubMed Journal
- 1992 Thomas, K. R., and Capecchi, M. R. (1987).
Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells.
Cell 51, 503-512. PubMed Journal
- 1993 Mansour, S. L., Thomas, K. R., and Capecchi,
M. R. (1988). Disruption of the proto-oncogene int-2 in mouse embryo-derived
stem cells: a general strategy for targeting mutations to non-selectable genes.
Nature 336, 348-352. PubMed Journal
- 1994 Gu, H., Marth, J. D., Orban, P. C., Mossmann,
H., and Rajewsky, K. (1994). Deletion of a DNA polymerase beta gene segment
in T cells using cell type-specific gene targeting. Science 265, 103-106.
PubMed
- 1995 Moon, A. M., Boulet, A. M., and Capecchi,
M. R. (2000). Normal limb development in conditional mutants of Fgf4. Development 127,
989-996. PubMed Journal
- 1996 Moon, A. M. and Capecchi, M. R. (2000). Fgf8
is required for outgrowth and patterning of the limbs. Nat. Genet. 26,
455-459. PubMed Journal
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©Jones and Bartlett Publishers (2007)
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