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Published December 11, 2014
Targeted Mutagenesis of Mouse Developmental Genes
Kirk R. Thomas
Howard Hughes Medical Institute, University of Utah, Salt Lake City 84112
ABSTRACT
Targeted mutagenesis of genes in
mouse embryo-derived stem ( E S ) cells provides a
means t o systematically alter the genome of a
mammalian organism. This technology permits the in
vivo mutagenesis of genes whose role in development
has heretofore been merely the subject of speculation.
Gene targeting in ES cells is mediated via a cellular
homologous recombination machinery. Parameters
that optimize the detection of homologous recombinants have been developed by targeting mutations
t o the Hprt gene. These gene-targeting protocols have
been used to demonstrate the requirement in mouse
development of one gene, the proto-oncogene Wnt-1.
Mice homozygous for null mutations in Wnt-1 show
severe abnormalities in the cerebellum and midbrain
and generally, but not always, die shortly after birth.
Key Words: Mice, Gene Targeting, Embryo-Derived Stem Cells, Embryonic Development
J. Anim. Sci. 1994. 72(Suppl. 3):1-8
A typical mammalian cell contains approximately
of the expression of
this complex genome is largely responsible for the
ordered progression seen during the development of a
single fertilized egg to an adult organism containing
perhaps 1014 cells. The elucidation of the specific
developmental roles for each of these individual genes
can come from several experimental sources. For
example, studies of transcriptional or translational
expression patterns lead to inference of the position of
specific genes in the developmental pathway. Alternatively, the role of a given gene may be defined by
analyzing the development of animals defective in
that gene. The characterization of mutant phenotypes
not only exposes the temporal and spatial position of a
gene’s action, but may lead to a functional understanding of the gene product a t the biochemical level.
Traditionally, developmentally important genes
have been characterized by the recognition of an
abnormal phenotype followed by identification of the
gene and the mutation responsible for that phenotype.
Although this approach has uncovered a number of
important processes in organisms as varied as bacteria and humans, its utility generally decreases with
the increasing complexity of the organism to be
studied. In the case of mammals, for example,
limitations imposed by generation time, mutation
frequency, diploidy, and internal embryonic development have severely limited the number and type of
spontaneously arising developmental mutations.
Thus, among the nearly 1,000 known mice mutants,
the class of recessive, embryonic lethal mutations is
severely underrepresented (Lyon and Searle, 1989).
Recent advances in cellular and molecular biology,
however, now permit the investigator to avoid the
limitations imposed by life history and generate
predesigned mutations in a model experimental mammal, the mouse. Although the prospect of systematically mutating each of the 100,000 mouse genes and
then assessing the developmental consequences of
these mutations is quite daunting, a short-list of
candidate developmental genes can be constructed.
Such genes may include those encoding growth
factors, proto-oncogenes, or genes whose homologues
are known to function developmentally in other
organisms. Because a large number of these potential
“developmental genes” now exist as cloned laboratory
reagents, they can be mutated in vitro, and targeted
back to the host organism (Capecchi, 1989). This
paper will include a discussion of the technology that
now permits the modification of virtually any mouse
gene and will discuss a specific example of a targeted
mutation that alters the normal murine developmental pathway.
lo5 genes. The precise regulation
Mutagenesis by Gene Targeting
The targeted mutagenesis of a gene is a multi-step
process. A copy of the gene of interest is first cloned
and mutated in vitro and is then introduced as a
“targeting vector“ into the host cell. Once inside the
cell, the mutagenicity of the targeting vector depends
on the homologous recombination machinery of its
host. This machinery, undoubtedly a component of a
DNA-repair pathway, can catalyze the exchange of
genetic information between two homologous pieces of
DNA. If this exchange involves the mutated sequence
and its chromosomal, wild-type homologue, a genetic
alteration can be fixed in the genome.
1
2
THOMAS
An extremely efficient homologous recombination
machinery exists in a variety of single-cell organisms,
such that the precise genomic modification of simple
eukaryotes such as fungi and certain protozoa has
become routine (Hinnen et al., 1978; Losanne and
Spudich, 1987; Cmz and Beverley, 1990; Ten-Asbroek
et al., 1990). The genome of higher eukaryotes,
however, has not been as tractable to manipulation.
One reason for this is the relative inefficiency of the
mammalian homologous recombination machinery. A
targeting vector introduced into a cultured mammalian cell, for example, has a very low chance of
interacting with its endogenous homologue. Rather,
exogenous DNA is integrated into the host genome at
apparently random (i .e., sequence-independent) chromosomal locations. The non-homologous reaction between exogenous and endogenous DNAs can be from
lo2 to lo4 more frequent than the homologous
interaction (Thomas et al., 1986; Thomas and Capecchi, 1987).
An additional obstacle to a useful mutagenesis
protocol is that the desired modification should ideally
be delivered to the germ line of the host organism. In
the case of the mouse, there are a limited number of
options for such delivery. One method takes advantage
of viral vectors to introduce DNA to early-stage
embryos (Jaenisch, 1976). Similarly, DNA injected
directly into the pronuclei of newly fertilized eggs will
be stably incorporated into the genome and thus
populate all derivative cells in the ensuing adult. The
low frequency of homologous recombination limits the
use of these methods and, in fact, only a single
instance of homologous recombination has been
reported following the injection of DNA into a
fertilized mouse egg (Brinster et al., 1989). To obtain
this single recombinant, 10,000 eggs were injected and
implanted and nearly 2,000 progeny were then
analyzed at the DNA level. An alternative strategy is
to introduce the targeting vector into embryo-derived
stem ( ES) cells (Thomas and Capecchi, 1987; Capecchi, 1989). Although these cells also exhibit low
recombination frequencies, their use reduces the
screening of recombinants from the animal to the cell
culture level. These cells, derived from pre-implantation-stage mouse blastocysts, can be grown and
manipulated in culture (Bradley et al., 1984; Gossler
et al., 1986; Robertson et al., 1986). Following their
transformation by exogenous DNA, these cells can be
screened and the appropriate recombinants cloned and
transferred to another blastocyst. The ES cells can
then colonize the host embryo and their progenitors
can contribute to all cell types in the developing
mouse, including germ cells. Thus, a mutation generated in vitro can be propagated indefinitely. For all
practical purposes, gene targeting in mouse ES cells is
the only currently available route for predetermined
modification of endogenous genes in a mammalian
genome.
Optimization of Gene Targeting
Although ES cells clearly contain the capacity t o
mediate homologous recombination, the low frequency
of this recombination creates a logistical problem in
isolating recombinants from a pool containing nontransformed cells and non-homologous recombinants.
Two basic approaches have been explored to overcome
this obstacle. One is to increase the ratio of homologous vs non-homologous recombinants; the other is to
specifically enrich for the growth of the homologous
recombinants.
The optimization of the experimental parameters
used for gene targeting in ES cells has been largely
done by mutating the Hprt gene (Thomas and
Capecchi, 1987; Doetschman et al., 1988; Deng and
Capecchi, 1992; Thomas et al., 1992; Deng et al.,
1993). This gene, encoding the enzyme hypoxanthine
phosphoribosyltransferase, has a number of qualities
making it the model gene. First, the gene is not
required for cellular viability. Second, cells carrying
mutations in Hprt are resistant to the purine analogue
6 thioguanine ( 6-TG), and can thus be selected for in
culture. Finally, the Hprt gene resides on the Xchromosome, such that in male-derived cells, only a
single mutational event is required to render a cell
hprt- (and thus 6-TW).
The most commonly used gene targeting strategy is
the replacement of sequences at the target locus with
sequences from the targeting vector. A typical gene
replacement at the Hprt locus is illustrated in Figure
1. The vector contains sequences homologous to the
target gene that have been mutated by the presence of
a selectable genetic marker (in this case the neor gene
from E. coli) inserted within the protein coding region
of the Hprt gene. This additional gene, mor, not only
serves to disrupt the target gene, rendering it inactive,
but provides a marker for the selection of cells
transformed by the targeting vector. Because most
transformation protocols are quite inefficient, the vast
majority of cells exposed to the targeting vector will
not incorporate it. By tagging the vector with a
marker gene, only those cells stably incorporating the
vector will survive the screen for positive targeting
events.
A typical targeting experiment at Hprt proceeds as
follows. The vector is introduced, generally by electroporation (Neumann et al., 1982; Chu et al., 1987),
into a population of ES cells; the cells are divided into
three aliquots grown under three growth conditions:
1) nonselectively, to assess the number of viable cells;
2 ) in the presence of G418, to assess the number of
cells stably transformed with the vector; and 3 ) in
media containing G418 and 6-TG, to select for those
cells containing an Hprt gene disrupted by the neo'
gene. This latter event can be confirmed at the gene
level, by performing Southern transfer analysis on
DNA extracted from G418', 6-Tff, cells. Typically, in
a targeting experiment at the Hprt locus, there is one
TARGETED MUTAGENESIS IN MURINE GENES
ne0
A
-4-Hx
x
I
I
I
I
I
I
1
1
1
2
3
4
5
6
I
I
I
I
I
I
1
2
3
4
5
6
I
I
I
I
7/a
9
6
neo
II
9
1 kb
Figure 1. Targeted mutagenesis by sequence replacement of the Hprt locus. (A) Pairing of a targeting vector
(top line) with the endogenous Hprt locus (bottom line).
The vector contains DNA sequences from the murine
Hprt locus in which the 7th and 8th exons have been
replaced with the neor gene. Following recombination at
positions indicated by X, endogenous sequences will be
replaced by vector sequences. (B) The hprt- locus,
resulting from homologous recombination as depicted
in ( A ) . Thin lines represent introns and flanking
sequences; solid boxes represent Hprt exons from 1 to
9; stippled boxes represent the mor gene.
G41ar colony for every 100 cells surviving transformation (Thomas and Capecchi, 1987; Deng and Capecchi,
1992). Of those G41W colonies, 1 in 1,000 is also
resistant to 6-TG. In all cases, those cells resistant to
both drugs contain the predesigned mutation of the
Hprt gene. Under optimum conditions, if one begins
with lo5 cells, one can obtain one cell with the desired
mutation.
Because the frequency of homologous recombination
depends on the enzymatic apparatus within the target
cell, there are a limited number of parameters that
can be vaned experimentally. Those parameters most
easily varied involve manipulation of the targeting
vector. Of the many variables in vector structure and
topology that have been tested, only three seem to be
of any consequence vis-&vis targeting frequency.
First, the vector must be linear. The recombination
machinery will apparently not recognize closed circular DNA (Thomas et al., 1986). Second, the DNA in
the vector must be highly homologous to the target
DNA. Mismatches of a few percent will severely
decrease recombination, such that DNA from different
mouse strains is not always recombinogenic (Deng
and Capecchi, 1992; TeRiele et al., 1992). Third, the
frequency of targeted recombination increases
logarithmically with linear increases in the length of
shared homology between vector and target. A twofold
increase in homology can result in a 10- to
20-fold increase in targeting frequency (Thomas and
3
Capecchi, 1987; Deng and Capecchi, 1992). Unfortunately, this increase reaches saturation at a vector
length of 12 to 15 kb of homology.
Identification and clonal isolation of cells carrying
targeted mutations at the Hprt locus present no severe
problems to the investigator. However, this same
luxury is not available to those interested in mutating
the vast majority of other genes. The mutant phenotype will most likely be unknown (hence the rationale
for performing the experiment), and may not be
manifest in cultured cells. Because of this, selection
protocols based on phenotype are difficult, if not
impossible, to design. The most straightforward
method for dealing with this obstacle is to screen for
the targeted mutation by examining the DNA of all
cells transformed with the targeting vector. This may,
however, entail the analysis of several hundred cell
lines, before the homologous recombinant can be
identified among the random integrant siblings. An
alternative is to reduce the number of nonhomologous
recombinants that must be analyzed. This can be
done, for example, by restricting the expressibility of
the positive selectable marker. By eliminating the
promoter sequences and(or) the 3' processing elements from the marker gene, the selectable marker
will only be active when inserted into another active
gene (Jasin and Berg, 1988). If the target gene is
expressed in ES cells, it meets that requirement, and
under such conditions, the ratio of uiable nonhomologoushomologous recombinants may be reduced
by over 50-fold.
A more versatile strategy, independent of the
expression of the target locus, has also been devised to
select against non-homologous recombinants. This
strategy, termed positive-negative selection, relies on
a difference in the exchange reactions mediating nonhomologous and homologous recombination (Mansour
et al., 1988). The former generally occurs through the
ends of the targeting vector, such that the entire
vector is inserted into the genome. If a linear targeting
vector contains on its ends DNA that is nonhomologous with the target, that DNA will be
incorporated. Homologous exchange, however, only
occurs over regions of homology, such that endterminal heterologous sequences will not be integrated. Thus, to decrease the viability of nonhomologous recombinants, a negative selectable
marker can be included at the e n d ( s >of the targeting
vector. Should the vector integrate by homology at the
target, the negative marker will be eliminated and the
cell remain viable. If the vector integrates at some
random position via its ends, the marker is retained
and the cell can be selected against. The most
commonly used negative selectable marker is the
thymidine kinase gene from the Herpes Simplex
Virus. The enzyme encoded by this gene can convert a
number of nucleotide analogues to products that are
toxic to most mammalian cells, including mouse ES
cells. Use of the positive-negative selection protocol
4
THOMAS
neo
1
2
3
4
1 kb
Figure 2. Mutant wnt-1- locus generated by gene
targeting. The neoTgene has been inserted by homologous recombination into the 2nd exon of the Wnt-1
gene. Thin lines represent introns, solid boxes represent
exons, stippled box represents the neo' gene.
can enhance the ratio of homologoudnon-homologous
recombinants by up t o 1,000-fold.
Use of the positive-negative selection protocol has
made it feasible to target mutations to virtually any
gene in the mouse. Genes whose function is merely a
matter of speculation can now be altered and the
consequences of this alteration examined at the
organismal level. This technology is particularly
amenable to the study of genes presumed to be
involved in development. Even if the gene is essential
to the viability of the embryo, recessive alleles can be
carried in heterozygous form and homozygosity
created by a simple mating. The progeny from the
intercross of heterozygous parents can then be examined for the mutant phenotype at all stages of
embryogenesis.
Targeted Mutagenesis of the Wnt-1 Gene
The list of potential mammalian developmental
genes is vast. It includes homologues of genes required
for development of other organisms, genes encoding
proteins known to act in tissue-specific fashion, genes
whose mRNA product is developmentally regulated,
and proto-oncogenes whose mis-expression or mutation affects growth and(or) differentiation. An example of one such gene, the indictment of which preceded
by several years its conviction, is the Wnt-1 (Int-1)
gene (reviewed in Nusse and Varmus, 1992). Identified first as an oncogene (Nusse and Varmus, 19821,
which when ectopically induced by proviral insertion
resulted in murine mammary carcinogenesis, the Wnt1 gene was later shown t o possess all the hallmarks of
a candidate development gene. It is a member of a
large gene family, consisting of 10 characterized
relatives expressed in mice (Gavin et al., 1990; Nusse
et al., 1991; Roelink and Nusse, 1991) and numerous
homologues in species ranging from C. elegans to H.
sapiens. The Drosophila Wnt-1 homologue is the
wingEess (wg) gene, a well-Characterized member of
the segment polarity class of genes and essential for
normal development of the fruit fly (Morata and
Lawrence, 1977; Rijsewijk et al., 1987). Furthermore,
in mice, expression of the Wnt-1 is restricted develop-
mentally to testes of adults and to dorsal midline cells
in the developing neural tube (Jakobovits et al., 1986;
Shackelford and Varmus, 1987; Wilkinson et al.,
1987). Based on this restricted pattern of expression,
it had been postulated that the Wnt-1 gene could be an
essential component in nervous system development.
This prediction has now been verified following the
generation and analysis of mice carrying mutations in
the Wnt-1 gene (McMahon and Bradley, 1990; Thomas and Capecchi, 1990).
A mutation in the Wnt-I gene was created by use of
the positive-negative selection strategy to introduce
the 1-kb n e d gene into the second protein-coding exon
of one Wnt-I allele in a mouse ES cell line. The site of
this insertion mutation is between codon 54 and 55 of
the 370-codon open-reading frame of the Wnt-I gene
and is predicted t o cause a complete loss of function of
the gene (see Figure 2). Embryo-derived stem cells
heterozygous for the mutation were injected into wildtype mouse blastocysts that were then allowed to
develop to term. One of the resulting chimeras was
able to pass the mutation through the germ line and
was used as a founder to establish a colony of mice
heterozygous for the Wnt-I mutation. The phenotype
of mice lacking a functional Wnt-1 gene is then
ascertained following the mating of Wnt-1+/writ-1animals. The genotypes of progeny from such matings
can be determined by DNA analysis of samples taken
from both embryonic and adult tissue and correlated
with the histological profile of the same individuals.
One of the first questions asked following such a
mating is whether the Wnt-I gene is required for
viability of the embryo. In the analysis shown in Table
1, the number of embryos and newborn pups of each of
the three possible genotypes is recorded as a function
of days in utero. k o m a total of 30 litters, containing
223 progeny, the wild-type ( +/+)/heterozygote ( +/-I/
mutant ( -/-I ratio is remarkably close to the predicted
Mendelian frequency of 1/2/1, suggesting nearly
universal survival of embryos. It should be noted that
two of the mutant fetuses examined at day 17.5 in
Table 1. Genotype of embryos from
heterozygous matings
Days in utero
No. of embryos of each genotype
(no. of litters)
9.5 (2)
10.5 (2)
11.5 (2)
12.5 (2)
13.5 (3)
14.5 (1)
17.5 ( 5 )
Newborn ( 13)
Total (30)
~
~
aIncludes two dead.
2
6
3
1
5
3
13
23
56
6
12
11
11
12
3
18
43
116
6
0
4
5
5
2
ga
20
51
TARGETED MUTAGENESIS IN MURINE GENES
Table 2. Viability of offspring from
heterozygous matings
Unanswered Questions
No. of animals of each genotype
~~
Day of death
0-.5
+I+
.5-1.5
3
1
1.5-2.5
> 30
17
2
5
~
+/-
-I-
4
5
0
34
14
4
1
1
utero were dead, although no other obviously dead
embryos were detected. In spite of the fact the
homozygous mutants are generally viable throughout
embryogenesis, they are frequently unable to survive
to weaning age. As seen in Table 2, of 20 newborn
animals genotyped as wnt-1-/wnt-l-, the vast majority
died immediately following birth, and only one individual survived to adulthood (after additional matings, another wnt-I- adult has survived; see Thomas
et al., 1991). Although this indicates some variability
in expressivity of the wnt-l- mutation, it also identifies the Wnt-1 gene as an important component of
normal mouse development.
The one wnt-1-lwnt-1- offspring that sullrived to
adulthood proved quite informative with respect to
determining the site of action of the Wnt-1 gene
(Thomas and Capecchi, 1990). The mutant animal
showed a number of behavioral abnormalities, including ataxia, hypertonia, and circling movements, all of
which are indicative of cerebellar abnormalities. Upon
examination of mutant animals from embryonic d 9.5
t o adulthood, severe malformation of pre-cerebellar
and cerebellar tissue was evident. As an example of
this phenotype, sagittal sections from heterozygous
and homozygous mutant individuals from embryonic d
11 and postnatal d 30 are shown in Figure 3. At the
embryonic stage ( a and b ) the mutant shows severe
underdevelopment of the metencephalon, the region a t
the midbrain-hindbrain junction destined to become
cerebellum, compared to its sibling. By adulthood ( c
and d), this underdevelopment has been translated
into a complete lack of the anterior cerebellar folia as
well as midbrain hydroencephaly.
Although all wnt-1-lwnt-1- animals showed malformations of the cerebellum, the mutation is highly
variable in expressivity. Mutant individuals examined
at birth, for example, fall roughly into two classes of
equal number (Thomas et al., 1991). One-half of the
animals have no identifiable cerebellar tissue and
little of the characteristic midbrain nuclei, the superior and inferior colliculi. The other class have
clearly visible midbrain nuclei (although their position is displaced) as well as some cerebellar tissue.
One conclusion from this analysis is that the Wnt-I
gene is minimally essential for the normal development of the anterior cerebellar compartment.
This analysis identifies the stage of development
that requires the Wnt-1 gene but leads to a number of
interesting questions regarding the function of this
gene. These same questions arise frequently when one
analyzes other developmental mutants and thus
deserve some consideration. The first is, What is the
biochemical function of Wnt-1 gene product? Because
of the stage of the first identifiable phenotype,
embryonic d 9.5, a time of rapid proliferation of
neuronal cells and their precursors, it is tempting to
speculate that the wnt-I protein is required for the
expansion of cells destined to populate the anterior
cerebellum. The Wnt-I gene product has been shown
in culture (and suggested by analogy to its Drosophila
homologue, wg) to be a secreted protein (Heuvel et
al., 1989; Papkoff and Schryvre, 1990; Nusse and
Varmus, 1992), and is thus a candidate for a regionspecific mitogen. Such a designation awaits further
investigation.
A second question arising from this analysis is,
Why is the wnt-l- phenotype restricted t o the
cerebellar-midbrain region of the developing nervous
system when the gene is expressed over a much
greater portion of the dorsal neural tube (Wilkinson et
al., 1987)? A number of answers include: 1) it is
required elsewhere, but our detection techniques are
too insensitive; 2 ) it is required elsewhere, but in its
absence, other genes can compensate; the fact that
expression of other members of the Wnt-gene family do
overlap that of the Wnt-I gene makes them likely
candidates for such complementing activity (Gavin et
al., 1990); 3) expression must not necessarily correlate with gene function; assuming the Wnt-1 protein
interacts with other proteins, functional activity
depends on simultaneous expression of all interactive
components. It should be emphasized that this lack of
100% correlation between gene expression and mutant
phenotype is not restricted to wnt-1 mutants, but
extends to a number of other developmental genes as
well (Zijlstra et al., 1989; Koller et al., 1990; Soriano
et al., 1991; Donehower et al., 1992).
The final question posed by this analysis is that of
expressivity. In the case of wnt-I- mice, a variability
in expressivity is manifest in two forms: 1) viability,
ranging from death in utero t o survival to adulthood,
and 2 ) quantity of brain tissue, ranging from loss of
the anterior cerebellum to loss of the entire cerebellum
and some adjacent midbrain nuclei. In general there
are three explanations for variability in expressivity:
leakiness of the mutation, variable genetic background, and stochastic fluctuations in the activity of
other genes.
Partial activity of a mutant gene is always a
possibility, unless of course the mutation is a complete
deletion. In the case of the wnt-l- mutation, there are
two arguments to suggest that the mutation is a null,
loss-of-function allele. First, the position of the insertion of the mor gene should prohibit translation of the
6
THOMAS
a
+I
d
C
---
*--*
-1-
Figure 3. Sagittal sections of wnt-l- mice (a and b). At 11.5 d embryos were fixed in Bouin’s reagent, embedded in
paraffin, sectioned (10 p m ) ,and stained with hematoxylin and eosin (c and d). Brains were prepared as described
previously (Thomas and Capecchi, 1990). Mid-sagittal sections (10 pm) stained with hematoxylin and eosin are
shown. Genotypes were determined by analysis of DNA from either yolk sacs [a and b) or tails (c and d). met
metencephalon; cf = cranial flexure; cb cerebellum; arrowhead in a and b indicates metencephalic region and in
d the primary fissure. +theterozygous for wnt-1-; 7homozygous for wnt-1-.
-
-
carboxy-terminal 80% of the Wnt-1 protein. Based on
sequence analysis and comparisons with other Wnt1-gene family members, the untranslated portion of
the protein is predicted t o contain a significant
number of h c t i o n a l domains. The second evidence
against the partial activity explanation is that a
second, spontaneously occurring, mutant allele of wnt1 has recently been identified and shown also to vary
in expressivity (Thomas et al., 1991). This allele,
called swaying ( s w ) (Lane, 1967), contains a single
base pair deletion that is predicted to cause premature
termination of translation that would eliminate 50%
of the Wnt-2 protein. Not only is the phenotype of sw
homozygotes identical in all respects to that of the
targeted mutant, but the compound heterozygote,
containing both mutant alleles, exhibits the same
variability in expressivity as well.
The phenotypes caused by mutant alleles may be
influenced by the genetic background of the animals in
-
-
which they are expressed (Ramirez-Solis et al., 1993).
The generation of targeted mutants often involves the
outcrossing of two strains of mice, and the wnt-lallele has been maintained on a non-inbred background. To ascertain whether the extremes in phenotype could be explained by modifier loci present in one
or more of the founder lines, the wnt-l- mice were
crossed several times with different inbred and
outcrossed lines of mice. No evidence of specific
effector loci could be found (Thomas and Capecchi,
unpublished data). Such variable expressivity of a
mutant gene under a constant genetic background has
been seen in several other instances as well.
It seems likely, then, that the variability in
expressivity seen in this and other cases (Chisaka et
al., 1992;Mansour et al., 1993; Ramirez-Solis et al.,
1993) may be the result of random variations in
expression of genes involved in growth and development of tissues in the affected region. In the case of
TARGETED MUTAGENESIS IN MURINE GENES
the wnt-1- phenotype, for example, expansion of cells
required for the posterior cerebellum and midbrain,
the t w o structures variably affected, may involve two
redundant pathways. In the absence of one of these,
that containing Wnt-I, the other becomes the sole
avenue for development of those structures. If activity
of this other pathway is at or near some threshold
requirement, it is likely that on occasion sufficient
activity may not be provided. It is possible that such
alternative pathways may be uncovered by analysis of
mutations with phenotypes similar to those caused by
the absence of the Wnt-I gene.
The generation and analysis of mice deficient in the
Wnt-I gene have demonstrated the potential of gene
targeting in embryonic stem cells as a mechanism to
study mammalian development. Using an identical
approach, it now should be feasible t o mutate virtually
any gene in the mouse and t o ascertain the consequences of the mutation a t the organismal level.
Although this discussion has focused on loss-offunction mutations, this same technology can now be
expanded to introduce subtle mutations into target
genes. This should not only permit the investigator to
modify discrete domains in the gene of interest, but
should also allow the precise re-creation of specific
mutations identified in other species, for example,
those causing human disease.
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