Human Molecular Genetics, 2000, Vol. 9, No. 12 1843-1852
© 2000 Oxford University Press
Transplacental injection of somite-derived cells in mdx mouse embryos for the correction of dystrophin deficiency
1IRCCS Ospedale Maggiore Policlinico, Milan, Italy, 2Centro Dino Ferrari, Institute of Clinical Neurology, University of Milan, Milan, Italy, 3IRCCS Eugenio Medea, Bosisio Parini, Italy, 4Université Paris 7, Case 7136, 2 place Jussieu, 75251 Paris, France and 5Wohl Virion Centre, Windeyer Institute, University College London, 46 Cleveland Street, London W1P 6BD, UK
Received 29 March 2000; Revised and Accepted 12 May 2000.
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ABSTRACT |
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Duchenne muscular dystrophy (DMD) is a lethal recessive disease caused by the absence of dystrophin in skeletal muscle, heart and other tissues. No cure is available at present for DMD. Here we describe a new strategy for the correction of dystrophin deficiency based on the transplantation of normal somite-derived cells into mdx mouse embryos. Somite-derived cells were isolated from E11.5 transgenic mouse embryos expressing the LacZ gene under the control of the muscle-specific desmin promoter and injected into the uterine circulation of pregnant mdx mice at gestational days E11.5–E17. Approximately 30% of the injected mdx embryos survived the procedure. Donor somite-derived cells were able to cross the placenta and migrate into host embryonic tissues. The pattern of donor cell distribution in host tissues depended on the gestational age of the transplanted embryos. Cells were found in hindlimb muscles, diaphragm, heart and ribs in E11.5 treated embryos and in the skull, ribs, vertebrae and lung of E15–E17 treated embryos. Normal dystrophin transcripts were detected in muscle and bone by RT–PCR. Histochemical analysis showed co-localization of LacZ and dystrophin expression in 5% of soleus and quadriceps muscle fibres and in 4% of heart myocytes of two of seven 8-week-old treated mdx mice.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Duchenne muscular dystrophy (DMD) is an X-linked recessive lethal muscle wasting disease affecting 1 in 3500 boys (1). It is caused by mutations of the dystrophin gene and the consequent absence of dystrophin in skeletal muscle and other tissues (2). At present no cure is available for DMD. An experimental model of DMD is the mdx/mdx mouse, where absence of the membrane-associated protein dystrophin is caused by a point mutation (3). Using this model, different experimental therapies have been tried, including transplantation of myoblasts from normal donor mice (4). Transplanted mononuclear muscle precursor cells were shown to fuse with mature muscle myofibres of the dystrophic host to produce a mosaic expressing dystrophin. Despite the good efficiency of dystrophin gene transfer obtained in the mdx mouse model, myoblast transplantation in DMD patients has been disappointing (5,6). Very few myoblasts survived in the host muscles after transplantation and only a small proportion of the surviving cells could express dystrophin or its mRNA (7). This might have been due to inadequate immunosuppression or to the advanced state of the disease, which results in extensive muscle fibrosis and adipose substitution. The newly introduced myoblasts did not migrate to damaged areas, but remained relatively close to the site of injection. The large number of myoblasts required to treat all of the affected muscles as well as the inability of the transplanted cells to migrate from the injection site reduce the likelihood that this method will be successful in treating DMD (5,6).
Recently, considerable effort has focused on the isolation of a population of multipotential muscle stem cells that could be transplanted into dystrophic mdx and human skeletal muscle and repopulate the diseased tissue. Interestingly, bone marrow transplantation in myelo-ablated mice resulted not only in myeloid reconsitution but also in some transplanted cells participating in myogenesis, suggesting that a population of transplanted bone marrow-derived stem cells had myogenic potential (8). As such, bone marrow transplantation has been performed to correct dystrophin deficiency in the mdx mouse, though only a few fibres became dystrophin-positive after transplantation (9).
Somites are mesodermal structures that appear transiently along each side of the neural tube during the development of vertebrates and are responsible for the formation of muscles (10,11). Ventrally, the somites form the mesenchymal sclerotomes, which give rise to the vertebral column and ribs. Dorsally, cells form the medial part of the myotomes, which give rise to the epaxial musculature, fibroblasts and endothelial cells. The lateral lip of the dermomyotomes is the source of the hypaxial musculature (12). These lips migrate ventrally and form the body wall musculature. Muscle precursors also disperse and migrate into the somatopleura to form the appendicular musculature (13,14).
In this study we have used somite-derived cells isolated from normal mouse embryos to evaluate whether these cells are able to migrate, proliferate and participate in myogenesis when injected into mdx host embryos. Exploitation of the multipotency and migration ability of somite-derived cells may result in correction of dystrophin deficiency at an early embryonic stage and, potentially, in long-term therapy of DMD. Indeed, haematopoietic stem cells have been successfully transplanted in utero into human (15) and mouse (16,17) fetuses resulting in reconstitution of the haematopoietic system for the treatment of X-linked severe combined immunodeficiency. We have developed a new method to introduce somite-derived cells into mouse embryos via the uterine circulation. We have used somites from transgenic embryos carrying a reporter LacZ gene to follow the fate of injected cells. The LacZ gene was under the control of regulatory elements of the desmin (des) gene, which is expressed in skeletal muscle and heart (18). Somites were obtained from the thoracic and lumbar regions of embryonic day (E) 11.5 embryos of transgenic mice and injected into the uterine circulation of pregnant mdx mice on gestational days E11.5–E17. The resulting offspring were analysed for the presence of ß-galactosidase (ß-gal)-positive cells and dystrophin-positive fibres to trace the distribution of cells in the tissues.
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RESULTS |
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Somite-derived cells cultured for 48 h express muscle-specific markers
Cell migration from the somites starts at E9.5 and is almost completed at E11.5 (19,20). In preliminary experiments we obtained similar results using somitic cells from E9.5 and E11.5 donor embryos. We chose donor embryos at E11.5 as they were easier to manipulate and clearly expressed the LacZ gene under control of the des promoter. Histochemical staining of whole donor embryos showed nuclear expression of ß-gal in the somites and heart, consistent with muscle-specific transcription of the LacZ gene driven by the des promoter (Fig. 1a). Donor embryos were dissociated and the cells obtained from the somites were plated on dishes coated with collagen, gelatin and laminin and cultured for 48 h. Approximately 90% of the isolated cells removed from the thoracic and mid-lumbar regions of E11.5 donor embryos were viable at the time of plating, as judged by trypan blue exclusion. None of the cultures contained neurons or notochord cells. Approximately 50% of the total cultured cells showed nuclear ß-gal expression (Fig. 1b). Their myogenic phenotype was also demonstrated by desmin immunostaining (data not shown). Approximately 30% of the cultured cells were slightly elongated and stained positive for myosin heavy chain. These data suggest that a large proportion of the cells had acquired the capacity for terminal myogenic differentiation after cultivation in vitro for 48 h. Such cells were injected into the uterine circulation of wild-type and mdx/mdx host mothers.
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Post-surgical follow-up
Of the mdx/mdx embryos, 82 were injected with donor cells at the early E11.5 stage and 27 at the E15–E17 stage. Of the wild-type embryos, 210 were injected at the E11.5 stage and 178 at the E15–E17 stage. Approximately 70% of the mdx/mdx and 50% of the wild-type embryos died during the first day of treatment due to haemorrhaging. Mortality was higher after the early (E11.5) treatment than after the late (E15–E17) treatment. Overall, the wild-type embryos were more resistent to surgery and recovered better than the mdx/mdx mice. The treatment seemed to have no harmful effect on development of the embryos that survived. Viability of the treated embryos was assessed by the presence of a visibly beating heart and movement of the embryo. Embryo size and gross morphology were also compared with those of age-matched, untreated controls. Newborn mice were active, appeared to be healthy and were nursed by their mothers. We examined a total (wild-type and mdx/mdx) of 233 fetuses, 52 newborns and 7 adult mice, but found no abnormality in either fetuses or adults (Table 1).
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Stage-related ability of somite-derived cells to invade embryos
To determine the timing of migration and the distribution of des–LacZ cells, a total of 215 wild-type and 18 mdx E11.5 embryos were analysed 4, 12, 24 and 48 h after injection by 5-bromo-4-chloro-3-indolyl-ß-galactopyranoside (X-gal) staining of whole embryos. Uninjected embryos were used as negative controls (Fig. 2). The placenta gave a non-specific reaction that prevented visualization of the specific blue nuclear staining of the donor cells. Placenta invasion was therefore monitored by PCR only and transgenic donor cells were found in the placenta up to 48 h after injection. ß-gal expression was detected in the umbilical cord 12 h after injection (Fig. 2d and e). ß-gal-positive cells were also detected in several regions of the embryos, such as the head and umbical and limb buds, 24 h after treatment (Fig. 2b). Treated embryos showed ß-gal expression in the four limb buds 48 h after cell injection (Fig. 2c and f). Donor somite-derived cells in the mdx/mdx recipient embryos were also detected by PCR amplification of both the normal dystrophin gene and the des–LacZ transgene. PCR of des–lacZ gave a specific band of 450 bp. Amplification of the endogenous des gene (350 bp band) was used as an internal control. Normal and mutant dystrophin genes were identified by MaeIII digestion of the PCR product. Normal dystrophin gave a 150 bp band plus two 25 bp fragments whereas the mutated dystrophin gave a 150 bp band plus a single 50 bp fragment. Treated E11.5 mdx/mdx embryos analysed 48 h after injection contained the normal dystrophin gene and the des–LacZ transgene in several tissues. Figure 3 shows the results obtained in the forelimb, intercostal and dorsal neck muscles, diaphragm and ribs. Somite-derived cells did not invade the muscles of the limb and trunk of embryos transplanted on E15 and E17, although a positive reaction was found in the diaphragm of some E15 embryos. Treated E15 and E17 embryos analysed 48 h after injection contained donor cells in the skull, ribs, limb bones, vertebrae, lung and diaphragm. No specific PCR band was observed in tissues from 19 non-transplanted embryos. Thus, cultured somite cells seemed to invade the muscle tissues best in early (E11.5) embryos. Table 2 summarizes the results obtained in 10 mdx/mdx embryos injected on E11.5 and in 8 mdx/mdx embryos injected on E15–E17. Interestingly, donor cells were also found in the lung and liver of the mothers (Fig. 3).
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Distribution of donor cells in adult mice
A similar pattern of donor cell distribution was found in adult mice derived from embryos injected on E11.5 and E15–E17. Ten neonates from different mdx foster mothers were obtained and seven of them were kept until they were 2 months old. PCR analysis of various tissues from these mice indicated that donor cell distribution varied according to the gestational age of the recipient (Table 3). Donor cells were found in the hindlimb soleus and gastrocnemius muscles of three of four mice injected on E11.5; two of these mice also had donor cells in the heart. Lungs and bones were the only non-muscle tissues found to be PCR-positive in some mice (Table 3). Mice injected on E15–E17 had donor cells in the bones, diaphragm and brain but not in hindlimb muscles (Table 3). Tissues from untreated mice scored negative.
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Detection of LacZ and dystrophin transcripts in treated mice
To detect dystrophin and LacZ mRNAs, RT–PCR analysis was performed on seven 2-month-old mdx mice which were injected with somite-derived cells on E11.5. Tissues which were shown by standard PCR to contain donor cells were evaluated for des–LacZ and dystrophin transcripts. Total RNA extracted from untreated and treated mdx mice was used as template for RT–PCR. Digestion of the dystrophin RT–PCR reaction products by MaeIII allowed normal and mutated dystrophin mRNAs to be distinguished. The normal dystrophin gene gave a 207 bp band plus two 25 bp fragments whereas the mutated dystrophin gave a 207 bp band plus a single 50 bp fragment. Normal dystrophin and LacZ mRNAs were found in the diaphragm, extensor digitorum longus and triceps brachii of two treated mice (Table 3 and Fig. 4), suggesting that transplanted somite-derived cells may participate in myogenesis. However, transcription of the LacZ and dystrophin genes was not totally superimposable. Dystrophin mRNA was also found in ribs, skull, humerus and femur, in which LacZ mRNA was undetectable (Fig. 4 and Table 4). Neither LacZ nor dystrophin transcriptional activity was detected in the other five 2-month-old mice (Table 3).
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Co-localization of dystrophin and LacZ expression
To identify which cell types were able to transcribe normal dystrophin and LacZ mRNAs, we performed histochemical staining on cryostat sections of muscles, brain, liver, lungs, kidneys and bones. ß-gal-positive nuclei were detected only in striated muscles (quadriceps and soleus) and in the heart. Positive LacZ myofibres were found in restricted areas corresponding to the soleus muscle of one mdx mouse and in the quadriceps distalis of the other mdx mouse. Muscle fibres scoring positive had peripheral nuclei and fibre diameter varied from 20 to 60 µm. In the soleus we counted 60 different fibres having ß-gal-positive nuclei, corresponding to ~6% of total fibres per cross-sectional area, and in the quadriceps distalis we counted 95 positive fibres, corresponding to 4% of total fibres per cross-sectional area. Transgene expression was also found in 3.7% of myocytes of the right atrium of the heart in one mouse. The pattern of LacZ gene expression was compared with that of dystrophin. Typical immunofluorescence for dystrophin was found in the same fibres that contained ß-gal-positive nuclei (Figs 5–7). The soleus contained dystrophin-positive fibres (54–76 fibres/section, mean 65), whereas the quadriceps distalis had 96–119 dystrophin-positive fibres/section (mean 106) and the heart had 102–117 dystrophin-positive fibres/section (mean 115). Dystrophin-positive fibres accounted for 6% of the cross-sectional area of the soleus, 4.2% of the quadriceps distalis and 4.5% of the heart. Control mdx muscles contained rare revertant fibres that were positive for dystrophin (21). The dystrophin antibody did not cross-react with autosomal dystrophin-related proteins such as utrophin. Positive fibres always seemed to exist as small groups, suggesting that there might have been clonal proliferation of a donor cell contributing to formation of the fibres in that region of the muscle.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have developed a new strategy to restore dystrophin expression in mdx mouse muscle based on transplantation of normal somite-derived cells in utero. Transgene DNA was found in wild-type and mdx/mdx embryos within 48 h of injecting somite-derived cells (Table 2 and Fig. 3), demonstrating transplacental passage through the circulatory pathway. Somite-derived cells crossed the placental barrier at a time during ontogeny when the natural homing sites were readily available, leading to adult mice with mosaics in a limited number of tissues. Muscle tissues could become colonized only when early-stage (E11.5) host embryos were injected, suggesting that the signals governing somite cell migration into developing muscles may be expressed transiently. Thus, accurate timing may be essential to achieve good efficiencies of transplantation. The precise pathway used by transplanted cells to home to host tissues is not known; cells might have used newly formed blood vessels or might have actively migrated from distant sites. Data from avian embryos suggest that blood vessels serve as routes for the migration of myoblasts into the limbs (22).
The fate of injected cells was evaluated by keeping seven neonates from six mdx/mdx foster mothers until they were 2 months old. We found LacZ and normal dystrophin gene transcription and expression in muscle and heart of mice treated on E11.5. Unexpectedly, RT–PCR analysis revealed the presence of dystrophin mRNA, but not LacZ mRNA, in the bones. The reasons for this discrepancy in transcription of the two genes are not clear; the des and dystrophin promoters may respond in a different way to environmental stimuli or non-myogenic cell types might have colonized the bones. Histochemical analysis of muscle and heart showed co-localization of dystrophin and ß-gal. Double positive fibres were generally grouped in certain segments of the muscles and always seemed to exist in small groups, suggesting that there might have been later clonal proliferation of a donor cell contributing to the formation of the muscle. These fibres were peripherally nucleated, indicating that they had not undergone regeneration in 2-month-old mdx mice, presumably because dystrophin expression protected these fibres from degeneration. Thus, somite-derived cells transplanted into a pre-immune fetus with congenital dystrophinopathy partly restored normal dystrophin production in striated muscles and heart during post-natal life.
Two major problems were encountered in this study: a high mortality rate of transplanted embryos and low levels of engraftment. The early gestation fetus is immunologically immature and uniquely tolerant to foreign antigens, allowing acceptance of allogeneic or xenogeneic cells without the need for immunosuppression. Moreover, the genetic background of all three types of mouse used (wild-type, mdx and transgenic) was C57BL/10 with haplotype H2b, which should exclude MHC-based immune rejection. We believe that the higher mortality rate observed early after transplantation in mdx/mdx embryos was due to the fact that they did not tolerate the mechanical stress involved in surgery. However, donor cells were also found in the mothers following intra-uterine injection of somite-derived cells which might have caused maternal immune responses against the transgenes at later stages. Further experiments are needed before any conclusions can be drawn on the reasons for the high mortality rate of transplanted embryos.
The efficiency of muscle engraftment depended on the gestational age of the transplanted embryos. It is conceivable that engraftment efficiencies may also depend on the developmental stage of the donor cells and that a more precise matching between the gestational age of donor cells and recipient embryos may increase the efficiency of the procedure. Moreover, in vitro manipulation of somite-derived cells may also adversely affect their differentiation ability. Only 50% of injected cells were committed to myogenic differentiation, as judged by ß-gal expression after cultivation in vitro. Thus, the precise nature of the cells contributing to muscle and bone formation in transplanted mice remains undetermined. It is conceivable that selection of subpopulations of such cells may increase the efficiency of engraftment.
Despite its present limitations, we believe that transplacental somite cell transplantation is a promising approach for the treatment of congenital disorders that can be diagnosed early in gestation. It may prevent irreversible pathological damage and allow engraftment in many distant areas affected by the disease. It would also be important to know whether somite cell xenotransplantation is a practicable alternative.
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MATERIALS AND METHODS |
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Desmin–LacZ transgenic mice
Transgenic mice specifically expressing the reporter gene LacZ in skeletal muscle (des–nlsLacZ) have been described previously (23). Briefly, the Escherichia coli LacZ gene carrying a nuclear localization signal was inserted in-frame into the first exon of the desmin gene. Founder animals were identified by PCR analysis of tail DNA using primers specific for the des–lacZ transgene. Primers sequences were as follows: Des1 (sense), 5'-TTGGGGTCGCTGCGGTCTAGCC-3'; Des1R (antisense), 5'-GGTCGTCTATCAGGTTGTCACG-3'; LacZ430R (antisense), 5'-GATCGATCTCGCCATACAGCGC-3'. These primers yield a product of ~450 bp for the LacZ transgene and a supplementary 350 bp band for the desmin gene. Homozygous male transgenic mice were bred by backcrossing with C57BL/10J females. Thus all experiments were performed with hemizygous embryos which expressed the LacZ transgene (Fig. 1).
Isolation of donor cells
E11.5 mouse embryos were obtained from des–nlsLacZ pregnant females. The embryos were removed from the decidua and the trunk, initially comprising axial structures, including notochord and neural tube as well as paraxial mesoderm. Somites were dissected using sharpened tungsten knives. Tissues were cut into small fragments and the neural tube and notochord complex were separated from somites by treatment with 0.1% collagenase (Sigma, St Louis, MO), 0.8% trypsin (Gibco BRL, Grand Island, NY) for 10 min at 37°C, washed in complete medium (see below) and then gently pipetted through a siliconized capillary to obtain a single cell suspension (19). Before injection, cells from somites V–XV were plated onto 60 mm collagen-coated dishes for 48 h. All cultures were grown in Ham’s F10 medium (Gibco BRL) supplemented with 15% fetal bovine serum (Gibco BRL) and gentamycin (50 µg/ml).
Somite-derived cell transplantation into embryos
Pregnancy was timed by checking for vaginal plugs. The day a plug formed was counted as E0. Approximately 106 cells were injected into C57BL/10J and C57BL/10ScSn mdx/mdx pregnant females when embryos were at E11.5, E15 and E17. The number of injected embryos was: E11.5, 210 embryos, E15, 88 embryos and E17, 90 embryos for wild-type mice; E11.5, 82 embryos, E15, 15 embryos and E17, 12 embryos for mdx mice. Pregnant mice were anaesthetized by i.p. injection of physiological saline (10 ml/kg) containing ketamine (5 mg/ml) and xylazine (1 mg/ml) and a limited low midline laparotomy performed. The uterine horns containing the embryos were exposed and cells were injected via 0.20 mm diameter needles, inserted into the uterine continuation of the medial femoral circumflex veins. The needles were connected to a peristaltic pump by heparinized Tygon tubes (Ika Labortechnik, Staufen, Germany). Each Tygon tube was connected to a sterile Eppendorf tube containing 5 x 105 cells/50 µl. Cells were delivered by laminar flow (4 µl/s) over a period of 13 s. The blood flow in the ovarian vessels was stopped before and during injection by pulling on traction threads placed around these vessels (Fig. 8). We checked that Reichert’s membrane, the yolk sac and ectoplacental cone were intact. There was no visible damage to the vessel wall during or after operation. The body wall muscle was closed with sutures and the skin with surgical staples.
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Detection of the dystrophin, ß-actin and des–LacZ genes
The brain, lungs, diaphragm, liver, kidneys, heart, bones and muscles were taken from the mice and divided in two identical halves. One half was used to isolate genomic DNA and total RNA, the other half was cryostat sectioned and used for X-gal histochemistry and immunodetection of dystrophin. Genomic DNA was isolated as described previously (24). The following primers were used to detect the normal and mutant dystrophin genes by PCR: dystrophin forward, 5'-CTCTGCAAAGTCTTTGAAAGAGTAA-3'; dystrophin reverse complementary, 5'-GAAGTTTATTCATATGTTCTTCTAGC-3'.
Dystrophin primers were located in exon 23 of the mouse dystrophin gene, which contains a point mutation in the mdx mouse (3). Sequence analysis of normal and mdx dystrophin mRNA confirmed the presence of a single base substitution at position 3185 in exon 23 of mdx mice. The primer dystrophin forward was designed to introduce a new site for the restriction enzyme MaeIII in the sequence of normal dystrophin cDNA, but not in the mutated cDNA of mdx mice (25). An initial denaturation at 94°C for 5 min was followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 45 s and extension at 72°C for 30 s, with a final extension at 72°C for 10 min. The 200 bp PCR product was extracted with phenol–chloroform, precipitated with ethanol, resuspended in 15 µl of H2O and incubated with MaeIII at 55°C for at least 5 h.
For RT–PCR total RNA was extracted using the RNAzol B isolation kit (Biotecx, Houston, TX) and was reverse transcribed for 1 h at 42°C using random hexamers and the GeneAmp RNA PCR kit (Perkin Elmer, Foster City, CA). Each amplification mix contained equal amounts of cDNA, 1.5 mM MgCl2, 30 pmol each primer, 5% dimethylsulphoxide, 200 mM each dNTP (Boehringer Mannheim, Mannheim, Germany) and 2 U Taq DNA polymerase (AmpliTaq; Perkin Elmer) in 1x PCR buffer (Perkin Elmer). Control reactions were performed in the absence of reverse transcriptase.
The following primers were used for RT–PCR: LacZ forward, 5'-GTCGTTTACAACGTCGTGACT-3'; LacZ reverse complementary, 5'-ATGGGCGCATCGTAACCGTGC-3'; ß-actin forward, 5'-TCCTGCGTCTGGACCTGG-3'; ß-actin reverse complementary, 5'-CCATCTCTTGCTCGAAGT-3'; dystrophin forward, 5'-CTCTGCAAAGTTCTTTGAAAGAGTAA-3'; dystrophin 2 reverse complementary, 5'-AACATCAACTTCAGCCATCCATTTC-3'.
Amplification parameters for LacZ and ß-actin are described in Russo et al. (26) and dystrophin PCR was performed as described above. PCR products were separated by electrophoresis on a 1% agarose, 2.5% NuSieve gel, stained with ethidium bromide and photographed under UV light. The 257 bp dystrophin PCR products were cleaned in a Microspin S-400 column (Amersham Pharmacia Biotech, Little Chalfont, UK), digested with MaeIII at 55°C for >5 h and separated on a 17.5% non-denaturing polyacrylamide gel.
Histochemistry and immunocytochemistry
Cultured des–LacZ cells were rinsed twice in phosphate-buffered saline (PBS), fixed in 0.25% glutharaldeyde in PBS and then incubated in 0.5 mg/ml X-gal solution for 3 h at 37°C. Blue cells were identified and counted by phase contrast light microscopy. To reveal ß-gal activity in whole embryos, 0.02% Nonidet P-40 and 0.01% sodium deoxycholate were added to the X-gal solution to enhance permeability of the tissues. Incubation was carried out for 10 h at 30 instead of 37°C to reduce background staining and minimize tissue damage (27). After staining, the embryos were fixed in 10% buffered formalin and paraffin embedded. For histochemistry on tissue sections, samples were frozen in liquid nitrogen-cooled isopentane and cryostat sectioned. Serial sections of different thickness were examined [12 µm for histochemical detection of ß-gal activity, 8 µm for immunohistochemical analysis and 8 µm for haematoxylin and eosin (H&E) staining]. Tissue sections were transferred to gelatin-coated glass slides and fixed by dipping the slides in a cold solution of 0.25% glutaraldehyde in PBS, pH 7.4, for 15 min, rinsed twice for 5 min in PBS and stained overnight at 32°C with 1 mg/ml X-gal solution. Slides were examined by light microscopy for ß-gal-positive myofibres (20). Immunohistochemical detection of dystrophin was performed with a polyclonal antibody against the 60 kDa C-terminal dystrophin fragment as previously described (28). LacZ- and dystrophin-positive myofibres were counted from adjacent H&E stained sections and expressed as a percentage of the total cryosectioned fibres. Thus, ß-gal and dystrophin percentage reliably reflected the ß-gal and dystrophin activity of the segment of muscle from which the sections were taken. For immunocytochemistry, cells were grown on glass coverslips until confluence, fixed in 4% paraformaldehyde in PBS for 15 min and permeabilized for 5 min with 0.5% Triton X-100 in PBS. Cells were then incubated with primary antibodies (MF 20 anti-myosin heavy chain and anti-desmin monoclonal antibodies; Novocastra, Newcastle, UK) overnight at 4°C. After washing with PBS, cells were incubated with FITC-conjugated goat anti-mouse IgG (Dako, Copenhagen, Denmark) for 1 h at room temperature and examined by epifluorescence microscopy.
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ACKNOWLEDGEMENTS |
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We thank R. Giorda, S. Livraghi and P. Ciscato for technical assistance. This work was supported by the Association Française contre les Myopathies and by the Centro Dino Ferrari, Institute of Clinical Neurology, University of Milan, Italy. A part of the work was funded by University Denis-Diderot Paris 7. A.F. is supported by the Wellcome Trust.
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FOOTNOTES |
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+ To whom correspondence should be addressed at: Institute of Clinical Neurology, University of Milan, Padiglione Ponti, Ospedale Policlinico, via Francesco Sforza 35, 20122 Milan, Italy. Tel: +39 02 5503 3817; Fax: +39 02 5519 0392; Email: gpcomi@unimi.it
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REFERENCES |
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1 Emery, A.E. (1989) Clinical and molecular studies in Duchenne muscular dystrophy. Prog. Clin. Biol. Res., 306, 15–28.[Medline]
2 Hoffman, E.P., Brown, R.H. and Kunkel, L.M. (1987) Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell, 51, 919.[Web of Science][Medline]
3 Sicinski, P., Geng, Y., Ryder-Cook, A.S., Barnard, E.A., Darlison, M.G. and Barnard, P.J. (1989) The molecular basis of muscular dystrophy in the mdx mouse: a point mutation. Science, 244, 1578–1580.
4 Partridge, T.A., Morgan, J.E., Coulton, G.R., Hoffmann, E.P. and Kunkel, L.M. (1989) Conversion of mdx myofibers from dystrophin-negative to positive by injection of normal myoblasts. Nature, 337, 176–179.[Medline]
5 Tremblay, J.P., Malouin, F., Roy, R., Huard, J., Bouchard, J.P., Satoh, A. and Richards, C.L. (1993) Results of a triple blind clinical study of myoblast transplantations without immunosuppressive treatment in young boys with Duchenne muscular dystrophy. Cell Transplant., 2, 99–112.[Web of Science][Medline]
6 Mendell, J.R., Kissel, J.T., Amato, A.A., King, W., Signore, L., Prior, T.W., Sahenk, Z., Benson, S., McAndrew, P.E. and Rice, R. (1995) Myoblast transfer in the treatment of Duchenne’s muscular dystrophy. N. Engl. J. Med., 28, 832–838.
7 Gussoni, E., Blau, H.M. and Kunkel, L.M. (1997) The fate of individual myoblasts after transplantation into muscles of DMD patients. Nature Med., 3, 970–977.[Web of Science][Medline]
8 Ferrari, G., Cusella-De Angelis, G., Coletta, M., Paolucci, E., Stornaiuolo, A., Cossu, G. and Mavilio, F. (1998) Muscle regeneration by bone marrow-derived myogenic progenitors. Science, 279, 1528–1530.
9 Gussoni, E., Soneoka, Y., Strickland, C.D., Buzney, E.A., Khan, M.K., Flint, A.F., Kunkel, L.M. and Mulligan, R.C. (1999) Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature, 401, 300–304.
10 Tam, P.P. and Beddington, R.S.P. (1986) Myogenesis. In Bellairs, R., Ede, A.A. and Lash, J.W (eds), Somites in Developing Embryos. Plenum Press, New York, NY, pp. 17–36.
11 Christ, B. and Ordhal, C.P. (1995) Early stages of chick somite development. Anat. Embryol. (Berl.), 191, 381–396.[Medline]
12 Christ, B., Jacob, M. and Jacob, H.J. (1983). On the origin and development of the ventrolateral abdominal muscles in the avian embryo. An experimental and ultrastructural study. Anat Embryol. (Berl.), 166, 87–101.[Medline]
13 Ordahl, C.P. and Le Douarin, N.M. (1992) Two myogenic lineages within the developing somite. Development, 114, 339–353.[Abstract]
14 Chevallier, A., Kieny, M. and Mauger, A. (1977) Limb–somite relationship: origin of the limb musculature. J. Embryol. Exp. Morphol., 41, 245–258.[Web of Science][Medline]
15 Touraine, J.L., Raudrant, D., Rebaud, A., Roncarolo, M.G., Laplace, S., Gebuhrer, L., Betuel, H., Frappaz, D., Freycon, F. and Zabot, M.T. (1992) In utero transplantation of stem cells in humans: immunological aspects and clinical follow-up of patients. Bone Marrow Transplant., 9, 121–126.
16 Pixley, J.S., Tavassoli, M., Zanjani, E.D., Shaft, D.M., Futamachi, K.J., Sauter, T., Tavassoli, A. and MacKintosh, F.R. (1994) Transplantation in utero of fetal human hematopoietic stem cells into mice results in hematopoietic chimerism. Pathobiology, 62, 238–244.[Web of Science][Medline]
17 Flake, A.W., Roncarolo, M.G., Puck, J.M., Almeida-Porada, G., Evans, M.I., Johnson, M.P., Abella, E.M., Harrison, D.D. and Zanjani, E.D. (1996) Treatment of X-linked severe combined immunodeficiency by in utero transplantation of paternal bone marrow. N. Engl. J. Med., 335, 1806–1810.
18 Li, Z., Mericskay, M., Agbulut, O., Butler-Browne, G., Carlsson, L., Thornell, L.E., Babinet, C. and Paulin, D. (1997) Desmin is essential for the tensile strength and integrity of myofibrils but not for myogenic commitment, differentiation, and fusion of skeletal muscle. J. Cell Biol., 139, 129–144.
19 Sze, L.Y., Lee, K.H., Webb, S.E., Li, Z.L. and Paulin, D. (1995) Migration of myogenic cells from the somites to the fore-limb buds of developing mouse embryos. Dev. Dyn., 203, 324–336.[Web of Science][Medline]
20 Li, Z.L., Marchand, P., Humbert, J., Babinet, C. and Paulin, D. (1993) Desmin sequence elements regulating skeletal muscle-specific expression in transgenic mice. Development, 117, 947–959.[Abstract]
21 Hoffman, E.P., Morgan, J.E., Watkins, S.C. and Partridge, T.A. (1990) Somatic reversion/suppression of the mouse mdx phenotype in vivo. J. Neurol. Sci., 99, 9–25.[Web of Science][Medline]
22 Solursh, M., Drake, C. and Meier, S. (1987) The migration of myogenic cells from the somites at the wing level in avian embryos. Dev. Biol., 121, 389–396.[Web of Science][Medline]
23 Li, Z.L. and Paulin, D. (1993) Different factors interact with myoblast-specific and myotube-specific enhancer regions of the human desmin gene. J. Biol. Chem., 268, 10403–10415.
24 Shrager, J.B., Naji, A., Kelly, A.M. and Stedman, H.H. (1992) A PCR-based assay for the wild-type dystrophin gene transferred into the mdx mouse. Muscle Nerve, 15, 1133–1137.[Web of Science][Medline]
25 Asselin, I., Tremblay, M., Vilquin, J.T., Guerette, B., Roy, R. and Tremblay, J.P. (1995) Quantification of normal dystrophin mRNA following myoblast transplantation in mdx mice. Muscle Nerve, 18, 980–986.[Web of Science][Medline]
26 Russo, S., Tomatis, D., Collo, G., Tarone, G. and Tatò, F. (1998) Myogenic conversion of NHI3T3 cells by exogenous MyoD family members: dissociation of terminal differentiation from myotube formation. J. Cell Sci., 111, 691–700.[Abstract]
27 Hogan, B., Beddington, R., Constantini, F. and Lacy, E. (1994) Manipulating the Mouse Embryo: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
28 Prelle, A., Moggio, M., Comi, G.P., Gallanti, A., Checcarelli, N., Bresolin, N., Ciscato, P., Fortunato, F. and Scarlato, G. (1992) Congenital myopathy associated with abnormal accumulation of desmin and dystrophin. Neuromusc. Disord., 2, 169–175.[Medline]
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