DEVELOPMENTAL DYNAMICS 212:229–241 (1998) Dmdmdx-bgeo: A New Allele for the Mouse Dystrophin Gene KARIN WERTZ* AND ERNST-MARTIN FÜCHTBAUER Max-Planck-Institut für Immunbiologie, Freiburg, Germany ABSTRACT 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 INTRODUCTION 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@ immunbio.mpg.de Received 4 July 1997; Accepted 23 October 1997 230 WERTZ AND FÜCHTBAUER 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 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 protein. 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 NEW MOUSE DYSTROPHIN ALLELE 231 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 232 WERTZ AND FÜCHTBAUER 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 RNA. 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). Phenotype 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 NEW MOUSE DYSTROPHIN ALLELE 233 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. 6C,D). DISCUSSION 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 234 WERTZ AND FÜCHTBAUER 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. NEW MOUSE DYSTROPHIN ALLELE 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 235 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. 236 WERTZ AND FÜCHTBAUER 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) mdx-bgeo 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. NEW MOUSE DYSTROPHIN ALLELE 237 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 esophagus. 238 WERTZ AND FÜCHTBAUER 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, 1996); 1993); 1996); 1996); NEW MOUSE DYSTROPHIN ALLELE 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. EXPERIMENTAL PROCEDURES 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). Genotyping 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- 239 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). RT-PCR 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). Immunoblotting 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). 240 WERTZ AND FÜCHTBAUER Histology 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. 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