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Dmdmdx-bgeo: A New Allele for the Mouse Dystrophin Gene
Max-Planck-Institut für Immunbiologie, Freiburg, Germany
During a gene trap screen, an
insertion of the gene trap vector into the dystrophin gene, creating a new allele for the Dmd gene,
has been discovered. Because the ROSAbgeo vector was used, the new allele is called Dmdmdx-bgeo.
The insertion occurred 38 of exon 63 of the dystrophin gene, resulting in a mutation that affects all
presently known dystrophin isoforms. In contrast to spontaneous or ENU-induced alleles,
Dmdmdx-bgeo can be used to follow dystrophin expression by staining for b-galactosidase activity.
The high sensitivity of this method revealed additional and earlier expression of dystrophin during embryogenesis than that seen previously with
other methods. Dystrophin promoters are active
predominantly in the dermamyotome, limb buds,
telencephalon, floor plate, eye, liver, pancreas
anlagen, and cardiovascular system. Adult
Dmdmdx-bgeo mice show reporter gene expression
in brain, eye, liver, pancreas, and lung. In skeletal
and heart muscle, b-galactosidase activity is not
detectable, confirming Western blot data that
indicate the absence of the mutant full-length
protein in these tissues. Hemizygous Dmdmdx-bgeo
mice show muscular dystrophy with degenerating muscle fibers, cellular infiltration, and regenerated muscle fibers that have centrally located
nuclei. Some mutant animals develop a dilated
esophagus, probably due to constriction by the
hypertrophic crura of the diaphragm. Dev. Dyn.
1998;212:229–241. r 1998 Wiley-Liss, Inc.
Key words: gene trap; dystrophin; Dp71; mutation; LacZ; Duchenne/Becker muscular dystrophy
Mutations in the human dystrophin gene are responsible for Duchenne and Becker muscular dystrophies
(DMD/BMD; Koenig et al., 1987). This X-linked disease
affects 1 in 3,500 live-born boys and is lethal before the
end of the third decade of life. The gene is encoded by
the largest gene locus detected to date. Spanning
approximately 2.5 Mb (den Dunnen et al., 1989) of
genomic DNA in humans, it covers 0.03% of the male
genome. The gene encodes several protein isoforms,
which are the results of transcription from at least
eight promoters as well as of alternative splicing and
polyadenylation (Blake et al., 1994; Nishio et al., 1994).
The four types of 14-kb, full-length mRNAs are tranr 1998 WILEY-LISS, INC.
scribed from individual promoters and differ in that
they have unique first exons (Klamut et al., 1990; Boyce
et al., 1991; Gorecki et al., 1992; Nishio et al., 1994).
The genomic organization of the dystrophin gene is
conserved between humans and mice. The full-length
protein has a calculated molecular mass of 427 kDa and
consists of four domains (Koenig et al., 1988). The
N-terminus serves as an actin-binding domain (Hemmings et al., 1992; Ervasti and Campbell, 1993). It is
followed by the rod domain (a stretch of 24 spectrin-like
repeats; Kahana et al., 1994), a cysteine-rich domain,
and the C-terminus (Koenig et al., 1988). The Cterminal region binds to syntrophins, whereas the
cystein-rich domain associates with b-dystroglycan
(Campbell and Kahl, 1989; Suzuki et al., 1994; Rafael et
al., 1996). b-dystroglycan, a transmembrane protein, is
associated with laminin by binding to a-dystroglycan
(Ibraghimov-Beskrovnaya et al., 1992). Thus, dystrophin can serve as a link between the cytoskeleton and
the extracellular matrix. Additional proteins, such as
sarcoglycans (a, b, g, d; Madvahan and Jarrett, 1995)
and dystrobrevins (Sadoulet-Puccio et al., 1996), are
also associated, forming the dystrophin-glycoprotein
complex (Ervasti and Campbell, 1991).
The absence of dystrophin or one of the sarcoglycans
or laminin leads to muscular dystrophy (Matsumura et
al., 1992; Roberds et al., 1994; Bönnemann et al., 1995;
Helbling-Leclerc et al., 1995; Lim et al., 1995; Nigro et
al., 1996; Noguchi et al., 1995). Therefore, it is assumed
that dystrophin forms a network beneath the cell
membrane, which, through anchorage to the dystrophinglycoprotein complex, provides a support, especially in
sensitive cells that are subjected to mechanical stress
(Menke and Jockusch, 1991) or hypoxic stress (Mehler
et al., 1992). However, the smaller dystrophin isoforms
lack the actin-binding domain. Therefore, the dystrophin gene products may have additional functions. The
shorter dystrophin versions are named according to
their molecular weight: Dp260, Dp140, Dp116, Dp71,
and Dp40. They consist of different numbers of repeats,
the cysteine-rich domain, and the C-terminal domain,
except that Dp40, which shares its promoter and Nterminus with Dp71, lacks part of the C-terminal
domain (Tinsley et al., 1993). Until now, no function
could be assigned to any of the short dystrophin isoforms. Certain mutations in the C-terminal region of
*Correspondence to Karin Wertz, Max-Planck-Institut für Immunbiologie, Stübeweg 51, D-79108 Freiburg, Germany. E-mail: wertz@
Received 4 July 1997; Accepted 23 October 1997
the gene in humans are associated with mental retardation in DMD patients. Thus, it has been suggested that
the shorter dystrophin versions play a role in cognition
(Rapaport et al., 1991; Comi et al., 1992; Bushby et al.,
1995; Lenk et al., 1993). The dystrophin isoforms are
expressed in patterns that are specific for each variant.
The muscle-type Dp427 is present in skeletal, cardiac,
and smooth muscle (Schofield et al., 1993, 1995). The
brain version of Dp427 is found in the cerebral cortex
and hippocampus (Gorecki et al., 1992), and the Purkinje isoform is produced in the cerebellar Purkinje
cells (Gorecki et al., 1992), whereas the L-type Dp427 is
expressed in lymphoblastoid cells (Nishio et al., 1994).
Dp260 is expressed specifically in the retina, Dp140 in
brain and embryonic kidney (Durbeej et al., 1997), and
Dp116 in Schwann cells (Byers et al., 1993). In contrast,
Dp71 expression is detected in many tissues (Lederfein
et al., 1992). Where it has been investigated, Dp40
expression coincides with Dp71, except that, in embryonic stem (ES) cells, only Dp40 is detected, and, in fetal
liver, only Dp71 is expressed (Tinsley et al., 1993).
Because the mutant protein for Dp40 in Dmdmdx-bgeo
would be indistinguishable from the mutant Dp71, we
will refer to the Dp40 and Dp71 mutant proteins
together simply as Dp71.
To date, five mouse alleles of dystrophin have been
isolated. The original Dmdmdx mouse, a spontaneous
mutant, bears a dystrophin gene with a point mutation
in exon 23 (Bulfield et al., 1984; Sicinski et al., 1989). In
addition, there are four ENU-induced alleles in the
mouse (Cox et al., 1993; Im et al., 1996). Hitherto, no
LacZ-knock-in has been reported.
Here, we describe a gene trap mutant mouse line in
which all dystrophin isoforms are mutated. Taking
advantage of the LacZ-reporter, we were able to detect
new aspects of Dmd gene expression during embryogenesis as well as in adult organs. Furthermore, the
muscular dystrophy phenotype of Dmdmdx-bgeo mice,
including pathologic alteration of the esophagus, is
described. This phenotype can be explained by the
absence of a detectable level of full-length dystrophin
protein in skeletal muscle.
Results of the Gene Trap Screen
The 1,759 blastocysts injected with gene trap ES cells
gave rise to 276 male chimeras, 70 of which were proven
to transmit the ES cell genome to their offspring. These
represent 20 different gene trap events. In ten of these
mouse lines, we detected LacZ-expression at day 11.5 of
embryogenesis. Five of them expressed the trapped
gene already before implantation. Seven lines exhibited
widespread to ubiquitous expression. Three lines
showed a restricted expression pattern, and one of
these lines, which was derived from CJ7 cells, is
presented in this paper. Breeding data already suggested an X-chromosomal integration of the ROSAbgeo
gene trap vector.
Dystrophin Gene Is Disrupted
by the Vector Integration
Southern blot analysis using EcoRI, which cuts only
once in the gene trap vector, showed that the mouse line
possesses a single ROSAbgeo (Fig. 1A) integration site
in the genome (Fig. 2A). Northern blot analysis had
revealed that the fusion transcript is expressed in liver
(not shown); thus, a liver cDNA library was prepared
from an adult mutant male. Screening of this library
yielded two individual clones containing fusion transcripts identical in the extent of the 58 end. Five-prime
to the splice acceptor of bgeo, both sequences match
with the dystrophin Genbank sequence S62620 (Fig.
1C). Because this represents the sequence of the Dp71specific exon spliced to exon 63, the integration of
ROSAbgeo apparently took place 38 of exon 63 of the
dystrophin gene (Fig. 1B). To prove that the two clones
represent a true fusion RNA, which is indeed present in
the gene trap line, reverse transcriptase-polymerase
chain reaction (RT-PCR) was performed on embryonic
cDNA both from the dystrophin mutant and from
another unrelated gene trap line (Fig. 2B). Whereas
bgeo and Dp71 transcripts could be detected in both
gene trap lines, only Dmdmdx-bgeo RNA contained fusion
transcripts of Dp71 and bgeo. This confirms that dystrophin is the gene that is mutated in this mouse line.
Because the integration site is located downstream of
all known promoters of this gene, all transcript variants should be affected by the mutation. RT-PCR data
using primer pairs recognizing exon 62 and exon 64 or
bgeo demonstrate that, indeed, not only Dp71/Dp40 but
also longer dystrophin transcripts are mutated (Fig.
2C). However, a low amount of a PCR-product reflecting
wild-type mRNA could be amplified by RT-PCR from
brains of Dmdmdx-bgeo males. This suggests that, in some
cases, the transgenic bgeo exon is skipped by alternative splicing, but this seems to be a minor percentage.
Moreover, full-length wild-type dystrophin protein,
which is normally expressed in brain, muscle, or retina,
could not be detected in Western blots in these tissues
from mutant males (Fig. 3). Mutant full-length dystrophin is not found in these organs of Dmdmdx-bgeo males
either, which could be due to instability of transcript or
In wild-type liver, brain, and retina, Dp71 is present.
In the mutant tissues, in contrast, no Dp71 could be
detected using the anti-C-terminus antibody MANDRA 1.
This is consistent with the C-terminus of dystrophin
being replaced by bgeo in the mutant protein, and this
was also shown by immunostaining of identical Western blots with anti-b-galactosidase (b-gal) antibody
40–1a. The Dp71-bgeo fusion protein has a calculated
molecular mass of 149 kDa, 146 kDa of which are
encoded by bgeo. The anti-b-gal antibody recognizes a
triplet of bands of approximately 120–160 kDa in brain,
liver, and retina. Because the dystrophin-bgeo fusion
proteins have a molecular weight roughly 80 kDa
higher than the corresponding wild-type protein, the
Fig. 1. A: Gene trap vector ROSAbgeo. Gene trap cassette inserted
into pGen- vector in reverse orientation with respect to the retroviral
genome. SA, adenovirus-derived splice acceptor; bgeo, encodes bgalactosidase (b-gal)-neomycin phosphotransferase fusion protein; pA,
polyadenylation signal of bovine growth hormone gene. B: Schematic
drawing of splicing scenario in Dmdmdx-bgeo. C: cDNA sequence representing Dp71 fusion transcript. The sequence 58 of bgeo sequences matches
the Genbank sequence S62620, which corresponds to Dp71-specific
exon 1 and dystrophin exon 63.
Western signal probably derives solely from the Dp71/
Dp40-bgeo fusion protein. Consequently, at least in
adult tissues, the majority of the b-gal activity we found
most likely reflects Dp71/Dp40 expression. Due to the
high sensitivity of the X-Gal-staining procedure, it is
possible, however, that some of the LacZ expression we
can detect in tissues results from the presence of other
dystrophin-bgeo fusion proteins with levels of expression that are below the detection level in Western blots.
and diffuse (Fig. 4O). At 11.5 dpc, reporter gene expression in the limb is restricted to the limb bud center (Fig.
4L) and extends into the digits as they develop (Fig.
4O,P). At 14.5 dpc, staining in limb buds is found in
connective tissue and tendon primordia but not in
muscles or bones (Fig. 4S).
Expression in the cardiovascular system. At
9.5 dpc, the reporter gene starts to be expressed weakly
in the region around the developing atrioventricular
valves (Fig. 4C). In later stages, the epicardium and
cells of the ventricular septum are positive for reporter
activity as well (Fig. 4G,U). Starting from 10.5 dpc,
b-gal activity is found in the endothelium of largecaliber blood vessels (Fig. 4E), and it continues to be
present in adult vessels (e.g., in brain; see Fig. 5F).
Expression in the gastrointestinal and respiratory systems. In fetal liver, only large blood vessel
epithelia exhibit b-gal activity (Fig. 4W). Livers of adult
male Dmdmdx-bgeo mice express LacZ throughout in
hepatocytes, whereas, in heterozygous females, this
tissue shows a spotty staining (Fig. 5C). Epithelium of
embryonic and adult pancreas is positive for reporter
activity (Figs. 4V, 5D). The pancreas of heterozygous
females also exhibits a mosaic staining pattern. b-Gal
Embryonic Expression Pattern
Expression in somites and limbs. From 9 days
postcoitum (dpc) onward, LacZ expression is found in
the lateral somite, with greater staining in the posterior part of each segment (Fig. 4B,E,I,J,K). Half a day
later, stained cells are found scattered in the dorsal
mesenchyme of the limb buds. At 10.5 dpc, bgeo fusion
protein is strongly expressed in limb buds and dermamyotome (Fig. 4E). At 11.5 dpc, a new, superficial
staining domain in the dermis of the back can be
detected that has a striped appearance but no sharp
border of staining (Fig. 4L). Later, this expression,
which is not strictly left-right symmetrical, loses its
regular striped pattern and becomes more irregular
Fig. 2. A: Southern blot analysis of Dmdmdx-bgeo genomic DNA cut with
EcoRI and hybridized with bgeo. Because EcoRI cuts once within the
gene trap vector, the two bands indicate a single integration site of the
construct. B: Reverse transcriptase-polymerase chain reaction (RT-PCR)
on random-primed cDNA from Dmdmdx-bgeo and from an unrelated gene
trap line. Both Dp71 and bgeo transcripts were amplified from cDNA of
each gene trap line, but the Dp71bgeo fusion message was detected only
in Dmdmdx-bgeo. The RNA source for Dmdmdx-bgeo tissue was heterozygous,
11.5-day dpc embryos. The RNA source for the unrelated gene trap line
was heterozygous 12.5 dpc embryos. C: RT-PCR on random-primed
cDNA from Dmdmdx-bgeo male, from heterozygous female, and from
wild-type tissue. The primer pairs used amplify all wild-type or mutant
dystrophin transcripts except for Dp71/Dp40, which were analyzed by the
RT-PCR shown in B. The data indicate that bgeo is also spliced to longer
dystrophin transcripts and that all but a very minor fraction of dystrophin
message is spliced to bgeo in mutants. The RNA source for Dmdmdx-bgeo
male tissue was adult brain, 11.5 dpc embryos were the source for
heterozygous female RNA, and 9.5 dpc embryos the source for wild-type
activity is detected in both exocrine and endocrine
pancreas (not shown). In tooth buds (Fig. 4R; 14.5 dpc)
the fusion protein is found in the epithelium. In the
lung of 14.5 dpc embryos, lacZ is expressed in the
endothelium of blood vessels (Fig. 4T). In contrast, in
adult lung, the bronchial epithelium is positive for
reporter activity (Fig. 5B).
Expression in the nervous system and in sensory
organs. Starting at 8.5 dpc, dystrophin expression is
found in the neural plate caudal to the developing hind
brain (Fig. 4A). At 9 dpc, the telencephalon is strongly
positive (Fig. 4B), and, half a day later, b-gal activity is
found in the roof of the rhombencephalon, in the optic
vesicle, and in the floor plate (Fig. 4C,D). In heterozygous
female embryos, the floor plate expresses LacZ in a striped
pattern (Fig. 4N,H). Later in embryogenesis, the fusion
protein is present in the lens, the retina, and the inner ear
(Fig. 4F,M). The adult brain exhibits b-gal activity in the
cerebral cortex (Fig. 5A), in the hippocampus (predominantly in the dentate gyrus; Fig. 5A,E,H), in the cerebellar
cortex (Fig. 5A,F,E), and in the olfactory bulb (Fig. 5A).
Expression in the skin. From 12.5 dpc, the whisker
follicles express LacZ in an outer cell sheath (Fig. 4O).
Two days later, hair follicles throughout the body are
positive (Fig. 4X).
Dystrophin Expression on the RNA Level
Whole-mount in situ hybridization experiments confirmed most of the data obtained with histochemical
staining for b-gal activity (Fig. 4Y; 9.5 dpc), except that
the striped expression pattern in the dermis of the back
at day 11.5 could not be seen (not shown).
Among 246 animals derived from matings of heterozygous parents, 72% were transgenic. This is in good
agreement with the expected 75%, assuming that our
routine genotyping is optimized to avoid false-positive
results. The heterozygous and homozygous females and
the hemizygous males are viable, as fertile as wild-type
129/Sv mice, and reach normal age. Histological sections of Dmdmdx-bgeo skeletal muscle reveal muscular
hypertrophy and dystrophy with degenerating muscle
fibers, cellular infiltration, and regenerated muscle
Fig. 3. Western blots of adult Dmdmdx-bgeo and wild-type tissues
immunostained with antibodies against the dystrophin C-terminus or
b-gal. Full-length dystrophin protein is not detected in Dmdmdx-bgeo muscle,
brain, or retina, in contrast to wild-type tissues. C-terminus-specific
antibody MANDRA1 detected Dp71 in wild-type brain, liver, and retina but
not in the same tissues from the mutant. On the other hand, anti-b-gal
antibody 40–1a recognized a triplet of proteins in the mutant tissues. This
is consistent with the replacement of the C-terminus of dystrophin with
bgeo. The size of the b-gal-positive proteins suggests that they represent
the Dp71/Dp40-bgeo fusion transcript. No larger proteins could be
detected by anti-b-gal antibody.
fibers with centrally located nuclei in males (Fig. 6B).
Indirect immunofluorescence on cryosections using the
polyclonal antidystrophin antibody 6–10 shows a lack
of sarcolemmal staining (Fig. 6E,F). The tendinous part
of the diaphragm is extended at the expense of the
hypertrophic and fibrotic muscular parts. In 5 of 12
6- to 18-month old males, a megaesophagus (Fig. 7) was
found. It most likely develops because of a constriction
of the diaphragm by the hypertrophic crura, because no
alteration of the muscularis of the esophagus or stomach could be found in these animals. The weight of the
left heart ventricle in relation to the body weight is
increased in Dmdmdx-bgeo males by 19.6% compared with
wild-type animals [ratio of left ventricle (g) to body
weight (g); 0.0040 versus 0.0033 in wild-type]. If
the weight of the left heart ventricle is compared
with the tibia length as a criterion of stature, independent of the actual body weight, then the relative weight
of the left heart ventricle is increased by 39.4%
[ratio left ventricle (g) to tibia length (cm); 0.073 versus
0.052 in wild-type]. This evidence of cardiac hypertrophy is also confirmed by the histological findings.
Cardiomyocytes are enlarged and more densely packed
in mutant myocardium than in wild-type heart (Fig.
Dmdmdx-bgeo Is a New Allele for the Mouse
Dystrophin Gene
In the Dmdmdx-bgeo mouse, bgeo is spliced to dystrophin exon 63, replacing the sequences encoding the
cysteine-rich domain and the C-terminus (Fig. 1B).
This mutation affects all known isoforms and enables
us to follow the expression of proteins that are tagged
by b-gal activity as long as the resulting fusion transcripts and proteins are stable. All of the five Dmdmdx
mutant alleles reported to date are point mutations
resulting in premature stops and/or aberrant splicing
(Fig. 8; Sicinski et al., 1989; Cox et al., 1993; Im et al.,
1996). Among these, the Dmdmdx3cv allele is most similar to Dmdmdx-bgeo, because it carries a mutation in
intron 65 that creates a new splice donor site, which
results in a frame shift (Cox et al., 1993). Because the
Dmdmdx3cv mutation occurs downstream of the most
38-located promotor, it is used frequently to investigate
the function of short dystrophin isoforms (Greenberg et
Fig. 4. Embryonic expression pattern of dystrophin-bgeo fusion protein (A–X) and dystrophin RNA (Y). Expression pattern analysis by X-Gal
staining was done on heterozygous female embryos (A–X). X-Gal
staining appears pink in darkfield (G–K,M,R,S). A: Embryo at 8.5 dpc. B:
Embryo at 9 dpc. C: Embryo at 9.5 dpc, left side view. D: Embryo at 9.5
dpc, right side view. E: Embryo at 10.5 dpc. F–K: Sections of embryos at
10.5 dpc. F: Head. G: Heart, transverse section. H: Floor plate, including
the pituitary gland. I: Trunk, abdominal level. J: Trunk, hindlimb level. K:
Somites, frontal section. L: Embryo at 11.5 dpc. M,N: Sections of 11.5 dpc
embryos. M: Inner ear. N: Floor plate (razor-blade section). O: Embryo at
12.5 dpc. P: Embryo at 14.5 dpc. Q–W: Sections of embryos at 14.5 dpc.
Q: Whisker follicle. R: Tooth bud. S: Limb bud longitudinal section with
staining in the subcutaneous connective tissue. T: Lung. In the developing
lung, the endothelium of blood vessels expresses LacZ. In contrast, in the
adult, staining is found in the lung epithelium (cf. Fig. 5B). U: Heart. The
location of the b-gal-positive cells could indicate expression in Purkinje
fibers. In addition, the epicardium is positive. V: Pancreas; b-gal activity is
seen in developing exocrine tissue. W: Liver. The surrounding area of
large-caliber vessels is stained. X: Embryo at 15.5 dpc. Y: Whole-mount
in situ hybridization demonstrating consistency of dystrophin expression
on RNA level with b-gal expression in Dmdmdx-bgeo embryos (shown at 9.5
dpc). The riboprobe recognizes dystrophin exons 66–75.
Fig. 5. Reporter gene expression in adult organs monitored by X-Gal
staining. A: Vibratome section of a heterozygous female brain. B: Lung of
a male. C: Liver of a heterozygous female. D: Pancreas of a heterozygous
female (darkfield). E–H: Portions of the vibratome section shown in A and
additional brain cryosections (G,H) counterstained with eosin. E: Hippocampus. F,G: Cerebellum at the border between cerebellar cortex and the
medulla. H: Dentate gyrus.
Fig. 6. Dmdmdx-bgeo mice lack dystrophin at the sarcolemma and
exhibit muscular dystrophy and cardiac hypertrophy. A,B: Hematoxylin
and eosin (H1E) histology of wild-type (A) and Dmdmdx-bgeo (B) skeletal
muscle. C,D: H1E histology of wild-type (C) and Dmdmdx-bgeo (D)
al., 1996). For such studies, the b-gal reporter of Dmd
could be advantageous, because it can serve as a cellautonomous marker for dystrophin-deficient cells. In addition, experiments on X-chomosome inactivation could profit
from the use of b-gal as a cell lineage marker, which could
also aid investigations on the effect of the dystrophin
myocardium. E,F: Indirect immunofluorescence with antidystrophin antibody 6–10 on Dmdmdx-bgeo (F) and wild-type (E) TA muscle. Tissues are
from 6-month-old males.
mutation on X-chromosome inactivation bias or selection
for cells expressing the wild-type dystrophin (see, e.g.,
Bittner et al., 1997). Moreover, the expression of neo under
the control of the dystrophin promoters provides the prerequisite for selection with G418 for dystrophin-expressing
cells from various tissues.
Fig. 7. Megaesophagus found in some Dmdmdx-bgeo males, probably due to constriction by the hypertrophic
diaphragm. The arrowhead indicates the dilated esophagus. In wild-type animals, this organ is covered by the
lungs; therefore, it is not visible. h, Heart; lu, lung; d, diaphragm.
The Dmdmdx-bgeo mutant could be useful for developing gene therapy vectors. b-Gal is an endogenous
protein for these animals; thus, it does not provoke an
immune response (Wells et al., 1997), whereas the
absence of b-gal activity from skeletal muscle permits
the use of lacZ reporter genes. In addition, because the
Dmdmdx-bgeo allele is genotyped easily, mutants can be
detected well before the phenotype has developed.
For Dmdmdx3cv, it cannot be ruled out that there are
additional mutations close to the dystrophin gene that
could be responsible for the reduced fertility or other
aspects of the phenotype. Mice with the similar
Dmdmdx-bgeo mutation do not breed readily, but fertility
is not significantly different from wild-type mice with
the same background (129/Sv). Breeding this mutant
from 129/Sv onto the C57Bl/6 background is underway
to determine whether there is an additional effect of the
Dmdmdx-bgeo mutation on reproduction. The muscular dystrophy phenotype is comparable to that reported for the
other Dmdmdx alleles. However, in 5 of 12 mutant males, we
found a megaesophagus associated with a hypertrophy in
the muscular part of the diaphragm. This has not been
reported previously for any other Dmdmdx allele, whereas a
DMD case with an esophagus diverticulum and mild
esophageal dilatation has been described (Leon et al.,
1986), and upper gastrointestinal dysfunction is seen frequently in DMD patients (Barohn et al., 1988; Jaffe et al.,
1990). Twelve- to eighteen-month-old Dmdmdx mice reportedly have a fibrotic smooth muscle layer of the esophagus
and stomach (Lefaucheur and Sebille, 1996), which we
could not detect in the Dmdmdx-bgeo mice with the dilated
Fig. 8. Integration site of ROSAbgeo in the dystrophin gene compared
with the location of the different promoters (L, C, M, P, R, B3, S, and G)
and the previously available Dmdmdx-alleles. Numbers indicate exon
numbers (modified from Im et al., 1996). The Dmdmdx-alleles are mutated
as follows: Dmdmdx, point mutation in exon 23 (Sicinski et al., 1989);
Dmdmdx2cv, mutation in splice acceptor of exon 42 (Im et al.,
Dmdmdx3cv, mutant splice acceptor site in intron 65 (Cox et al.,
Dmdmdx4cv, premature stop mutation at base 7,916 (Im et al.,
Dmd mdx5cv, 53-base-pair deletion in exon 10 (Im et al.,
Dmdmdx-bgeo, transgene insertion 38 of exon 63.
LacZ Staining Pattern Correlates With In Situ
Hybridization Data but Reveals Additional Sites
of Expression
both of which have been found in epithelia (Lidov et al.,
1993, 1995; Durbeej et al., 1997). The fact that only
larger sized vessels exhibit b-gal activity, although the
endothelial layer is common to all vessels, might indicate a role of dystrophins in anchorage of the endothelium to the basal lamina of mechanically stressed
vessels (Ginjaar et al., 1995).
The patchy staining of the liver and pancreas in
heterozygous females is most likely the result of
X-chromosome inactivation, which might also explain
the striped LacZ expression in the floor plate of heterozygous female embryos, because these tissues are
stained homogeneously in male and homozygous female animals. Expression in the floor plate has been
reported for Dp71 (Schofield et al., 1995). Mosaic dystrophin expression in heterozygous Dmdmdx females has
also been detected in the cerebellum (Fig. 5; Huard et
al., 1992). As for the myotome staining, it remains to be
determined in the mutants whether the Dp427 fusion
proteins are made in the embryo but not in the adult.
Alternatively, the staining in the telencephalon could
reflect Dp71 expression. Dystrophin expression in the
roof of the rhombencephalon and in the optic vesicle has
not been detected previously. A strong in situ hybridization signal for full-length dystrophin was shown over
Rathke’s pouch (Houzelstein et al., 1992). In Dmdmdx-bgeo,
in contrast, the posterior part of the pituitary is stained
much more strongly. Dp71 has not been found in the
pituitary. The b-gal activity in the inner ear is consistent with reports of Dp116 (and Dp427) in the hair cells
of the adult organ of Corti (Dodson et al., 1995). For the
adult brain, preliminary X-Gal staining results correspond well with the data collected by in situ hybridization (Gorecki et al., 1991, 1992; Gorecki and Barnard,
1995). However, a detailed analysis of LacZ expression
Expression in muscle lineage is first found in the
lateral somite at day 9. It has been shown with in situ
hybridization that the full-length dystrophin message
is expressed in the myotome as well as in cardiac and
skeletal muscle, whereas expression in smooth muscle
is very weak (Houzelstein et al., 1992). However, in
Dmdmdx-bgeo mice, embryonic muscles do not express
LacZ after the dermamyotome stage, and, for adult
Dmdmdx-bgeo mice, Western blot data revealed that the
mutant equivalent of Dp427 is not present in muscle.
Because the full-length fusion message or protein seems
to be unstable, it is unlikely that this variant is
responsible for the b-gal activity in the dermamyotome.
More likely, an expression of Dp71 that has not been
seen previously is made visible by the reporter. The
high sensitivity might also explain the new expression
domains in the dermis of the back or in the connective
tissue in the limbs, tendon primordia, hair follicles, and
pancreatic epithelium. For the telencephalon, fulllength dystrophin transcription has been shown from
13 dpc onward (Houzelstein et al., 1992; Schofield et al.,
1995; Tennyson et al., 1996). The proposed instability of
the full-length fusion message or protein probably
explains the absence of b-gal activity in smooth muscle
and myocardium, where Dp427 was shown to be present (Houzelstein et al., 1992). LacZ expression is found
in a subset of cells, perhaps Dp71, which is also
synthesized in the heart (Muntoni et al., 1995). The
localization of the staining is compatible with dystrophin expression in Purkinje fibers (Ginjaar et al., 1995).
Similarly, we suspect that b-gal activity in the larger
caliber vessels reflects expression of Dp140 or Dp71,
in the brain remains to be undertaken. Considering the
Western blot results for Dp427, it was not expected that
the cerebral and cerebellar cortex would be stained,
because the full-length fusion proteins could not be
detected in Western blots of adult brain. Therefore, it is
likely that shorter isoforms are also expressed at these
sites but have not been detected previously. Whereas
the presence of Dp71 has been shown previously in
tooth buds and whisker follicles (Schofield et al., 1995),
no dystrophin expression has been found in other hair
follicles. Because Dmdmdx-bgeo is a mutation that affects
all splice variants of dystrophin, it will be interesting to
compare the expression pattern and phenotype with
those of a recently created Dp71-specific lacZ ‘‘knock in’’
(Nudel and Yaffe, personal communication). In summary, we have demonstrated that the new Dmdmdx-bgeo
allele can be an advantageous tool with which to study
the function of dystrophin during embryonic development and postnatal life.
Production of Gene Trap Mouse Lines
R1 and CJ7 ES cells were grown as described previously (Nagy et al., 1993; Swiatek and Gridley, 1993);
6 3 106 R1-cells and 14 3 106 CJ7-cells were infected
with the retroviral gene trap vector ROSAbgeo (Fig. 1A;
Friedrich and Soriano, 1991) in the presence of 6 µg
polybrene (Sigma, St. Louis, MO) at a multiplicity of
infection of 0.0025 (calculated as the probability for a
cell to become neomycin-resistant after infection). Beginning 1 day after infection, the cells were selected for
10 days in G418 (200 µg/ml active substance). In the
middle of the selection period, the cells in some wells
were trypsinized and sown on fresh feeder cells without
expanding the culture area. Finally, 16 CJ7 clones and
18 R1 and CJ7 pools were produced, each of the pools
consisting of ca. 160 ES-cell clones. Each pool was
injected into C57Bl/6 or (B6D2F1xB6D2F1) blastocysts
(Wertz and Füchtbauer, 1994).
DNA was isolated as described previously (Laird et
al., 1991). Individual mouse lines were identified by
Southern blotting (Sambrook et al., 1989). Genotyping
was carried out routinely by dot blotting using a BRL
dot-blot device (BRL-Life Technologies, Gaithersburg,
MD). Briefly, approximately 20 µg of tail DNA was
denatured in 0.4 M NaOH for 30 min at 37°C in 96-wellplates. Subsequently, the samples were transferred to
the dot blot apparatus containing a Hybond N1 membrane (Amersham, Braunschweig, Germany) that had
been soaked in 0.4 M NaOH previously. After 30 min,
the sample was filtered through the membrane by
vacuum. After ultraviolet cross linking (Stratalinker,
Stratagene, La Jolla, CA) and rinsing, the blot in 30
phosphate, pH 7, and 0.1% sodium dodecyl sulfate
(SDS), it was hybridized with the 4-kb Xho I-fragment
of pSAbgeo (Friedrich and Soriano, 1991) in Church
buffer at 65°C overnight (Sambrook et al., 1989). Wash-
ing conditions were 30 mM sodium phosphate, pH 7,
and 0.1% SDS at 65°C.
Construction of a cDNA Library
Total RNA was prepared by using RNAzol-B (Tel-Test,
Friendswood, TX). Subsequently, poly-A-RNA was isolated
from total RNA with Oligotex (Qiagen, Hilden, Germany).
Two micrograms of twice enriched poly-A-RNA were used
to construct a cDNA library using the Superscript plasmid
system (BRL-Life Technologies) with the following modifications: cDNA was randomly primed, ds-cDNA was ligated
to EcoRI linkers (Pharmacia, Uppsala, Sweden) and EcoRI
cloned into pT7T319U cloning vector. The library was
screened by using the adenovirus-derived sequences from
the 58 end of bgeo as a probe. These are the sequences from
the splice acceptor up to the ATG of bgeo. They were
amplified by PCR with the primer pair #3076 (58-CGGTTGAGGACAAACTCTTCG-CGGTCTTTC-38) and #1339 (58GGGATCCGCCATGTCACAGA-38).
Two micrograms poly-A-RNA were subjected to cDNA
synthesis by applying 75 ng of random hexamer primers and 300 U Superscript II (BRL-Life Technologies),
as recommended by the manufacturer. One-tenth of the
reaction was used as a template in the PCR. Cycling
conditions were 95°C for 30 sec, 60°C for 30 sec, and
72°C for 30 sec, 30 cycles; and 72°C for 7 min.
bgeo-cDNA was amplified with the primer pair described above (#3076 and #1339), Dp71 was amplified
with primers in Dp71-specific exon 1 (Dp71s: 58CTTACTCCTCCGCTCTAA-38) and in exon 63 (58CATTTTGGGGTGGTC-38). Fusion cDNA of Dp71 and
bgeo was amplified with Dp71s and #1339. Fusion
cDNAs of bgeo and dystrophin versions containing exon
62 were detected by using #1339 and dys-62s (58CCAAACAAAGTGCCCTAC-38). Wild-type message was
amplified with dys-62s and dys-64as (the latter recognizes exon 64 with 58-AGCAAAGGGCCTTCTGGA-38).
The dystrophin probe for in situ hybridization was
amplified with primers recognizing dystrophin exon 66
(58-CGGGACGAACAGGGAGGAT-38) versus exon 75
(58-GGAGAGGTGGGCATCATC-38) and cloned into TA
vector (Invitrogen, La Jolla, CA).
Proteins were separated electrophoretically by SDSpolacrylamide gel electrophoresis (SDS-PAGE) in 6% or
12% polyacrylamide gels and were transferred to Hybond C extra membrane in a semidry blotter (Bio-Rad,
Cambridge, MA). The blots were incubated with
MANDRA1 a monoclonal antibody recognizing the
C-terminus of dystrophin (D8043; Sigma, St. Louis,
MO) and anti-b-gal antibody 40–1a (Developmental
Studies Hybridoma Data Bank, University of Iowa).
Antibody binding was detected by using ECL (Amersham, Braunschweig, Germany).
Heterozygous female embryos were used for analysis
of embryonic expression patterns. Histochemical staining for b-gal activity was performed according to Beddington and Lawson (1990). Stained embryos were
embedded in methacrylate (Technovit 8100; HeraeusKulzer, Werheim, Germany) and sectioned at 8 µm. For
cryosections, tissues were quick frozen in melting isopentane by using a cork disc as a support. Sections were
made on a Leica Cryoxy (Heidelberg, Germany) at
225°C, transferred to glass slides, and air dried for
15–30 min. For b-gal histochemistry, sections were
fixed with 4% paraformaldehyde for 10 min. For indirect immunofluorescence, sections were blocked with
2% rabbit serum in phosphate-buffered saline (PBS) for
10 min and subsequently incubated with polyclonal
antibody 6–10 (Byers et al., 1993) diluted 1:300 in 2%
rabbit serum in PBS at 37°C for 1 hr. Secondary
antibody was antirabbit immunoglobulin-fluorescein
isothiocyanate conjugate (Dianova, Hamburg, Germany; 1:400 in 2% rabbit serum; 45 min at room
temperature). Hematoxylin and eosin staining was
performed according to standard protocols.
Whole-Mount In Situ Hybridization
Whole-mount in situ hybridization was carried out
essentially according to the method of Wilkinson (1992),
with the following modifications: Hybridization was at
65°C, posthybridization RNase-treatment was not performed, and the washing stringency was 2 3 SSC, 0.1%
Tween 20, and 50% formamide at 65°C.
We thank Tom Gridley for CJ7 cells; Andras Nagy,
Reka Nagy, and Wanda Abramow-Newerly for R1 cells;
Phil Soriano for the gene trap vector ROSAbgeo; Carsten
Bönnemann for 6–10 antibody; and Bernhard Herrmann, Heiner Schrewe, Darek Gorecki, and Christian
Holubarsch for discussions. We are especially grateful
to Nathalie Daigle for substantial participation in this
work, Eva Götz and Tina Engist for excellent technical
assistance, and Davor Solter and Rolf Kemler for
discussion and support.
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