Introduction

Muscular dystrophies are genetic diseases caused by mutations in genes encoding muscle proteins, leading to progressive muscle degeneration 1. Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy, characterized by widespread degeneration of the skeletal, respiratory, and cardiac muscles, resulting in disability and premature death. DMD is caused by mutation of the dystrophin gene (DMD), which resides on the X chromosome 1,2. Limb girdle muscular dystrophy 2D (LGMD2D) is a less common autosomal recessive form of muscular dystrophy resulting from mutation of the α-sarcoglycan gene (SGCA) on chromosome 17. The phenotype of this disease is variable, depending on the nature of the mutations involved, with severe forms being similar to DMD 1,3. Limb girdle muscular dystrophy 2B (LGMD2B) is another autosomal recessive form of muscular dystrophy resulting from mutation of the dysferlin gene (DYSF) on chromosome 2. LGMD2B leads to a later-onset, milder form of muscular dystrophy characterized by progressive degeneration of skeletal muscles and resulting disability 1,4.

Because of their monoallelic genetic basis, the addition of genes or cells that could provide the DMD, SGCA, or DYSF protein that is deficient in these disorders has the potential to be an effective treatment. Gene therapy strategies to introduce functional DMD, SGCA, or DYSF genes to diseased muscle fibers are attractive approaches currently under development5,6,7,8,9. Even so, gene therapy may have diminishing efficacy as patients progressively lose muscle fibers with time. Moreover, gene therapy strategies typically do not correct muscle stem cells 10. In these settings, cell therapy strategies designed to restore healthy muscle cells may have value. If healthy muscle progenitor cells were introduced and could fuse to existing deficient fibers, as well as form new muscle fibers, regeneration of muscle might occur 11,12. The well-established ability of satellite cells and their myoblast progeny to engraft in skeletal muscle 13,14 provides a basis for cell therapy strategies, as does the use of muscle progenitor cells derived from pluripotent stem cells and other cells 15,16,17,18,19,20,21,22,23,24,25,26,27.

Cell therapy strategies require transplantation of living cells, which may involve introduction of foreign genes into the recipient organism. Gene therapy strategies are also often assisted by inclusion of foreign marker genes, such as luciferase. To create mouse models that would be appropriate for such experiments, including the transplantation of human cells into mice, we took advantage of recently created mouse strains that are severely immune deficient, lacking B, T, and NK cells. The NRG strain background is useful for muscle studies, since it tolerates irradiation 28. Ionizing radiation is sometimes used in muscle regeneration studies to eliminate the endogenous host stem cells, known as satellite cells, thereby facilitating engraftment of donor cells 29. The development of gene and cell therapies is facilitated in the background of immune-deficient animals, by removing rejection barriers and allowing the use of foreign markers and cells, thus expanding the range of permissible experimental designs. This study provides three novel severely immune-deficient mouse muscular dystrophy models that will be useful in the development of successful gene and cell therapies. We also characterize the models to provide valuable baseline phenotypic information about them. Comparing the results between strains may elucidate further the phenotypic differences between these three forms of muscular dystrophy, although we note that since the strains are not fully inbred, differences in modifier genes may be present between them that could affect phenotype.

Materials and Methods

Ethics statement

The Stanford Administrative Panel on Laboratory Animal Care approved all procedures performed on animals in protocol number 15766, assurance number A3213-01. The Stanford Comparative Medicine program is accredited by the Assessment of Laboratory Animal Care International.

Mouse strains

NOD (NOD/ShiLtJ, 001976), NRG (NOD.Cj-Rag1tmMomIl2rγtm1Wjl/Szj, 007799) 28, mdx4Cv (B6Ros.Cg-Dmdmdx-4Cv/J, 002378) 30, and mdx/scid (B10ScSn.Cg-PrkdcscidDmdmdx/J) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Bl/AJ mice (B6.A-Dysfprmd/GeneJ, 012767) 6 were provided by the Jackson Laboratory from a stock maintained by the Jain Foundation. SGCA-null mice 3 were a kind gift from Kevin Campbell.

PCR analysis of genotypes

Genomic DNA was extracted from mouse ear punch using Wizard Genomic DNA Purification Kit according to manufacture’s instructions (Promega, Cat. A1120). IL2rγ and Rag1 regions were amplified from 100 ng of genomic DNA (5 min at 95°C; 30 sec at 94°C, 30 sec at 51°C (IL2rγ) and 52°C (Rag1), 30 sec at 72°C, 35 cycles; 5 min at 72°C). The primers used to detect wild-type or mutant alleles are published on The Jackson Laboratory website. The primer sequences IL2RG-Common-Fwd (5’-AAGAGATTACTTCTGGCTGTCAG-3’) and IL2RG-Wt-Rev (5’-CTCTGGGGTTTCTGGGG-3’) were used to detect IL2rγ wild-type. The primers IL2RG-Common-Fwd and IL2RG-Mut-Rev (5’-ATGCTCCAGACTGCCTTG-3’) were used to detect IL2rγ mutant. The primer sequences Rag1-wt-Fwd (5’-TCTGGACTTGCCTCCTCTGT-3’) and Rag1-Common-Rev (5’-CATTCCATCGCAAGACTCCT-3’) were used to detect Rag1 wild-type. The primers Rag1-Common-Rev and Rag1-Mut-Fwd (5’-TGGATGTGGAATGTGTGCGAG-3’) were used to detect Rag1 mutant.

The following primer sets were used to identify mutant muscle disease gene alleles: Dysfprmd (5’-TTCCTCTCTTGTCGGTCTAG-3’), (5’-CTTCACTGGGAAGTATGTCG-3’) and (5’-GCCTTGATCAGAGTAACTGTC-3’); mdx4Cv (5’-TCAAGAACAGCTGCAGAACAGGAGA-3’) and (5’-GGATTGCATCTACTGTGTGAGGACC-3’); and Sgca (5’-GCCAGAGGCCACTTGTGTAG-3’) and (5’-ACTCACCTACCACGCTCACC-3’). Expected fragment sizes are given in Fig. 1b & c, Fig. 2b & c,and Fig. 3b & c.

Immunohistochemistry

Muscles were harvested and snap frozen in OCT in liquid nitrogen. Serial 8 μm cryostat sections were obtained throughout the muscle. Sections were fixed in 2% PFA for 10 minutes and washed 3X in PBS with 2% triton (PBS-T) for 1-2 min. Sections were blocked in 5% donkey serum in PBS-T for 1 hour. Primary antibodies were prepared in block solution and incubated overnight at 4°C in a humid chamber. The next day, slides were washed 3 times with PBS and stained with secondary antibody for 1 hour at room temperature. Slides were washed with PBS 3 times and visualized. Antibodies used were: rabbit anti-dystrophin (15277, Abcam, Cambridge, UK), rabbit anti-dysferlin (NCL-Hamlet Novocastra, Leica), and rabbit anti-Sgca (189254, Abcam). Donkey anti-rabbit conjugated to Alexa 594 (A21207, Life Technologies) was used as secondary antibody.

Western blots

Muscle lysates were prepared with RIPA Buffer supplemented with HALT Protease Inhibitor Cocktail (Thermo Fisher Scientific 78430, Waltham, MA) according to the manufacturer’s protocol. The supernatant containing protein extract was denatured with Laemmli Sample Buffer (Bio-Rad 1610737, Hercules, CA) supplemented with 100 mM DTT. In each lane of a 10% TRIS-glycine SDS-PAGE gel (Bio-Rad), 25 μg protein extract was electrophoresed at 70 V in ice cold PAGE running buffer (0.1% SDS, 25mM Tris, 250mM glycine). Samples were transferred onto 0.45 μm PVDF membrane (Thermo Fisher) for 50 minutes at 100 mA at 4°C. Membranes were blocked in 0.2% BSA and 2% milk diluted in TBS with 0.5% Tween-20. Dystrophin, dysferlin, and Sgca detections were respectively achieved with a 1:1000 dilution of rabbit-anti-dystrophin (15277, Abcam), rabbit-anti-dysferlin (124684, Abcam), and rabbit-anti-SGCA (189254, Abcam). GAPDH was probed with a 1:5000 dilution of rabbit-anti-GAPDH (181602, Abcam) in blocking solution. Rabbit antibodies were probed with a 1:5000 dilution of goat-anti-rabbit IgG HRP secondary antibody (Thermo Fisher). Blots were developed in Clarify Western ECL Substrate according to manufacturer’s protocol (Bio-Rad) and imaged using the ChemiDoc Touch Imaging System (Bio-Rad).

Histological staining

Tibialis anterior (TA), quadriceps (Q), hamstring (H), and gastrocnemius (G) muscles from NRG, mdx4Cv/NRG, BlAJ/NRG, and Sgca/NRG mice were isolated and flash frozen in mounting media (OCT, Satura Finetek, Torrance, CA). Cryosections 12 um-thick were stained using hematoxylin and eosin (Sigma, St. Louis, MO) following the manufacturer’s specifications. All pictures were taken using a Zeiss Aptiskop microscope.

To evaluate the membrane permeability of muscle, mice were intraperitoneally injected with Evans Blue dye (EBD; Sigma; 100 ul of 1% EBD in PBS per 10 g body weight). The following day, muscles were flash-frozen and ground with a mortar and pestle. Following an overnight incubation in formamide, EBD was quantified in the supernatant by measuring light emission at 630 nm 31.

Hydroxyproline, which is a direct measure of the amount of collagen/gelatin, was used to determine the amount of fibrosis in whole muscle. Hydroxyproline was measured according the manufacturer’s protocol (Kit-555; BioVision, Milpitas, CA). Briefly, muscles were dissected and incubated in 6N HCl for 3 hours at 120°C. Small volumes of the hydrolysates were then incubated with the Chloramine T and p–dimethylaminobenzaldehyde (DMAB) reagents. The resulting product, a chromogen, was then measured at 560 nm.

Treadmill

To measure mouse fatigue, mice were run on a treadmill (IITC Model 800; IITC Life Science, Woodland Hills, CA) with a 10° uphill incline. An electric shock bar grid at the end of the tread delivered a mild shock to mice if they stopped running. The fatigue time was measured as the time a mouse could run before hitting the electric shock grid 5 times. During the first week, mice were acclimated 3 times (Monday, Wednesday, Friday) at 8 m/min for 3 min. The following week, mice were similarly run 3 times with the speed set at 10-18 m/min, with increments of 2 m/min. The time that a mouse hit the electric shock grid for the 5th time was recorded as the run length. For each mouse, the times of the 3 runs were averaged. Measurements were performed at the same time each day to reduce variability.

Statistical analyses

To determine statistical significance for two groups, comparisons were made using a Student’s t-test. Statistical analyses were performed using GraphPad Prism v.6 (GraphPad Software, La Jolla, CA, USA). A p-value < 0.05 was considered significant.

Results

Construction of immune-deficient mdx4Cv, Bl/AJ, and Sgca-null mouse models

Immune-deficient mouse models are useful in cell therapy studies since they allow transplantation of cells expressing markers that would otherwise cause rejection, preventing long-term engraftment. Such markers include species-specific antigens such as those present on human cells. Immune-deficient mouse models are also valuable in gene therapy studies, enabling the transfer of genes that might otherwise be immunogenic, including human therapeutic genes and bioluminescent and fluorescent marker genes. An immune-deficient mdx4Cv/NSG mouse line was previously constructed and has been valuable 32. On the other hand, the NSG line of mice is highly susceptible to radiation-induced damage because of mutation of the Prkc gene, which is necessary for double-strand break repair. Since irradiation is a frequently used technique for pre-conditioning skeletal muscle for cell engraftment 29, we used the NRG strain, which is less sensitive to radiation, permitting more flexibility on dosage28. Lymphocytes are eliminated in NRG mice because of a mutation in the recombination-activating gene (Rag1), which is required for VDJ recombination in lymphocytes and is immune specific 28. A different immune-deficient DMD model with similar properties, featuring a non-revertable dmd mutation and Rag2 and Il2rb mutant alleles has also been reported33.

We generated three lines of dystrophic, immune-compromised mice; one that lacks dystrophin, one that lacks dysferlin, and another that lacks α-sarcoglycan. To generate mdx4Cv/NRG mice, we combined null mutations in the genes encoding rag1, interleukin 2 receptor gamma subchain (IL2rγ), and the mdx4Cv mutation in dystrophin (dmdmdx4Cv) that introduces a premature stop codon in exon 53 of the dmd gene. The dmdmdx4Cv mutation has a 10-fold lower reversion frequency than the original mdx mutation 30,34Details of the crosses involved are shown in Fig. 1a. The dmdmdx4Cv mutation was monitored in crosses by using PCR (Fig. 1c). To initiate breeding, C57Bl/6/mdx4Cv/4Cv females were first outcrossed with NOD males. Female progeny were outcrossed to NOD males, and this cross was repeated three times, generating backcross generations N1, N2, and N3. N3 generation NOD/dmdmdx4Cv/+ females were outcrossed twice to NRG males to obtain backcross generations N4 and N5 dmdmdx4Cv/NRG mice. The mice progressively changed in coat color from black to white through the crosses. In addition to the dystrophic dmdmdx4Cv allele, Rag1 and Il2rg mutations were monitored by PCR and maintained beginning with the N4 generation (Fig. 1b). N5 generation NRG/dmdmdx4Cv/+ mice were intercrossed to obtain dmdmdx4Cv/NRG breeding pairs homozygous for the dmdmdx4Cv, Rag1, and Il2rγ mutations. These mice were used to propagate and maintain by inbreeding the line, designated mdx4Cv/NRG.

Figure 1

Fig. 1: Characterization of mdx4Cv/NRG mouse model.

(a) Breeding scheme for generation of the mdx4Cv/NRG mouse colony. (b,c) PCR analysis showing the fragments used for identification of the NRG and mdx4Cv genotypes. Sizes of diagnostic fragments are noted in basepairs (bp). (d) Immunohistochemistry for dystrophin in gastrocnemius muscle shows the absence of dystrophin in mdx4Cv/NRG compared to NRG. (e) Western blot illustrating absence of dystrophin in mdx4Cv/NRG.

To generate BlAJ/NRG and Sgca/NRG mice, a similar breeding scheme was followed, but incorporating the Dysfprmd mutation in dysferlin or the Sgca-/- mutation in α-sarcoglycan instead of mdx4Cv(Fig. 2a, 3a). The Dysfprmd mutation and Sgca-/- mutation were obtained respectively from BlA/J mice 6 and Sgca-null mice 3. Dysfprmd, Sgca, Rag1 and Il2rγ mutations were monitored in crosses by using PCR (Fig. 2b & c; Fig. 3b & c). To initiate breeding, B6.A-Dysfprmd/GeneJ (Bl/AJ) females were first outcrossed with NOD males, and subsequent crosses were similar to those described above for establishment of the mdx4Cv/NRG mouse colony, producing the BlAJ/NRG mouse line (Fig. 2a).A similar breeding scheme was used to obtain Sgca/NRG mice from Sgca-/- females (Fig. 3a).

Figure 2

Fig. 2: Characterization of BlAJ/NRG mouse model.

(a) Breeding scheme for generation of the BlAJ/NRG mouse colony. (b,c) PCR analysis showing the fragments used for identification of the NRG and Bl/AJ genotypes. Sizes of diagnostic fragments are noted in basepairs (bp). (d) Immunohistochemistry for dysferlin in gastrocnemius muscle of NRG and BlAJ/NRG demonstrated the absence of dysferlin in BlAJ/NRG. (e) Western blot illustrating absence of dysferlin in BlAJ/NRG.

Figure 3

Fig. 3: Characterization of Sgca/NRG mouse model.

(a) Breeding scheme for generation of the Sgca/NRG mouse colony. (b,c) PCR analysis showing the fragments used for identification of the NRG and Sgca-null genotypes. Sizes of diagnostic fragments are noted in basepairs (bp). (d) Immunohistochemistry for a-sarcoglycan in gastrocnemius muscle of NRGand Sgca/NRG demonstrated the absence of a-sarcoglycan in Sgca/NRG. (e) Western blot illustrating absence of a-sarcoglycan in Sgca/NRG.

Muscle studies

The muscle phenotype of mdx4Cv/NRG mice was assessed in 6 month-old animals. Muscle sections from mdx4Cv/NRG and NRG mice were stained with antibodies that bound specifically to dystrophin. As shown in Fig. 1d, dystrophin was present at the sarcolemma of all fibers in NRG muscle, but was completely absent in muscle from mdx4Cv/NRG (Fig. 1d), validating the absence of this membrane-associated muscle protein in the disease model mice. Western blots further substantiated the absence of dystrophin (Fig. 1e).

To evaluate the BlAJ/NRG mice, muscle sections from 6 month-old BlAJ/NRG mice and the NRG control were stained with antibodies that bound specifically to dysferlin. We observed dysferlin staining predominantly localized to the membrane in the NRG mice (Fig. 2d), but an absence of staining in the BlAJ/NRG mice (Fig. 2d), consistent with a null phenotype for dysferlin. Western blots for dysferlin from the two samples confirmed the absence of dysferlin in the BlAJ/NRG mice (Fig. 2e). Likewise, immunohistochemistry (Fig. 3d) and western blotting (Fig 3e) confirmed the absence of Sgca protein in the Sgca/NRG mouse model.

To analyze any associated muscle pathology, hematoxylin and eosin (H&E) staining was carried out on muscle sections from the three mouse models. As seen in Fig. 4a, the NRG muscle appeared normal, while the muscles from mdx4Cv/NRG, BlAJ/NRG and Sgca/NRG mouse showed some infiltration by mononuclear cells and greater variability in fiber size. The percentage of centronucleated fibers (CNF) in gastrocnemius, hamstring, and quadriceps muscles was counted in each of the three mouse models (Fig. 4b). The values were highest for the mdx4Cv/NRG and Sgca/NRG mice, but were also statistically significant for the BlAJ/NRG mice compared to NRG mice. In the BlAJ/NRG mice, a stronger effect was observed in hamstring muscle, compared to quadriceps and gastrocnemius, but all values were significantly elevated over the NRG control. However, CNF was significantly higher in mdx4Cv/NRG and Sgca/NRG mice relative to BlAJ/NRG mice.

Figure-4

Fig. 4: Hematoxylin and eosin staining and centronucleation.

(a) H&E staining in gastrocnemius muscle of NRG, BlAJ/NRG, Sgca/NRG, and mdx4Cv/NRG shows increases in the numbers of centronucleated fibers (CNF) in dystrophic mouse muscle. These data are quantified in (b). Scale bar = 75 um. Mice were six months old, and data are mean ± SEM with n = 4-9 with *p<0.05. G = gastrocnemius, Q = quadriceps and H = hamstring muscles.

Evans blue dye (EBD) staining was used to analyze membrane integrity. EBD is excluded from normal muscle fibers, but penetrates muscle fibers with perturbed membrane integrity, such as those from mdx4Cv mice 31, since the absence of dystrophin leads to disruption of the muscle membrane. After injection of EBD in our 6-month old mouse models, we evaluated the different amounts of EBD observed (Fig. 5a). All three of the mouse models demonstrated evidence of muscle pathology with EBD. In the gastrocnemius and quadriceps muscles, the mdx4Cv/NRG and Sgca/NRG mice both displayed significantly higher amounts of EBD relative to BlAJ/NRG and NRG controls (Fig. 5a). EBD in hamstring muscle was significantly higher in Scga/NRG than in the two other mouse models. The amount of EBD observed in BlAJ/NRG was significantly higher than in NRG mouse for hamstring muscle, but not for gastrocnemius and quadriceps muscle. We also noted an age-related increase in EBD in a study of BlAJ/NRG mice that ranged in age from 6-15 months; all three muscle groups showed significant elevations in EBD over age-matched NRG controls by 15 months of age (Fig. 6a).

Figure-5

Fig. 5: Evan’s blue dye, hydroxyproline, and treadmill studies.

(a) The three mouse strains are compared to NRG for penetration of Evan’s blue dye (EBD). mdx4Cv/NRG and Sgca/NRG mice showed elevated EBD values in all three muscle groups tested, whereas BlAJ/NRG mice exhibited greater EBD membrane permeabilization only in hamstring muscle compared to NRG mice. (b) Significant differences were observed in hydroxyproline deposits in gastrocnemius and hamstring muscles in mdx4Cv/NRG and Sgca/NRG mice, but not in quadriceps. BlAJ/NRG did not show significant increases at this age. (c) During treadmill exercise, BlAJ/NRG mice became exhausted significantly earlier than NRG mice, while mdx4Cv/NRG and Sgca/NRG mice became exhausted much earlier. Mice were six months old and data are mean ± SEM with n = 4-9, with *p<0.05. G = gastrocnemius, Q = quadriceps and H = hamstring muscles.

Furthermore, dystrophic mice have greater collagen deposition in muscle compared to wild-type mice3534. To characterize collagen deposition in our three mouse models, hydroxyproline of 6-month old mouse muscle was analyzed. We confirmed greater collagen deposition in the gastrocnemius and hamstring muscles of mdx4Cv/NRG mice compared to NRG mice (Fig. 5b). Collagen deposition in the Sgca/NRG mice was elevated to a similar degree (Fig. 5b). No significant difference of hydroxyproline was observed in quadriceps muscle between the three mouse models and the NRG control. The amount of hydroxyproline did not change between NRG and BlAJ/NRG mice in the six-month old mice (Fig. 5b). However, hydroxyproline was significantly elevated in the three muscles analyzed in older BlAJ/NRG mice (Fig. 6b), reflecting progressive pathology in the BlAJ/NRG animals.

Figure-6

Fig. 6: Characterization of aged BlAJ/NRG mouse model.

Whereas 6-month old BlAJ/NRG did not shown any statistical difference for EBD and hydroxyproline with NRG muscles, older BlAJ/NRG did. The amount of EBD (a) and hydroxyproline (b) were increased in the majority of the older BlAJ/NRG mouse muscles studied, compared to younger animals. Data are mean ± SEM with n = 3-6, with p<0.05. G = gastrocnemius, Q = quadriceps and H = hamstring muscles.

To evaluate muscle function, we used a treadmill assay. The mdx4Cv/NRG mice were able to run for dramatically less time than the wild-type NRG controls, confirming the functional deficit of the mdx4Cv/NRG mouse strain (Fig. 5c). For each of these assays, the well-established mdx/Scid strain was also evaluated, with the result that the new mdx4Cv/NRG mouse strain and mdx/Scid were similar in each case (data not shown). These results suggested that mdx4Cv/NRG mice were severely impaired in mobility and that placing the mdx4Cv mutation in the more severely immune-deficient background did not significantly alter disease pathology. The Sgca/NRG mouse showed similarly abbreviated treadmill times, compared to the NRG control, revealing a deficit in muscle function similar to that of mdx4Cv/NRG mice (Fig. 5c). We were also able to demonstrate a statistically significant deficit in the BlAJ/NRG mice with this assay (Fig.5c). This deficit was milder than that seen in mdx4Cv/NRG and Sgca/NRG mice, consistent with the milder phenotype of LGMD2B compared to DMD and severe forms of LGMD2D.

Discussion

Immune-deficient mouse models are valuable resources for cell and gene therapy studies of genetic diseases. Genetic models allow assessment of phenotypes and detection of improvements to phenotypes that may be provided by experimental therapies. Such therapies often involve the use of foreign cells and genes whose use would be hampered in immune-competent animals. For example, the transplantation of human cells and the transfer of foreign genes stimulate rapid immune responses that would preclude longer-term experiments, since treated cells would be eliminated by the immune system. In recent years, the development of more completely immune-deficient mice that lack B, T, and NK cells that are less leaky than previous models such as Scid, have allowed long-term engraftment of human cells in strains such as NSG and NRG 28.

Crossing such mice with disease model mice remains a time-consuming and labor-intensive process. For that reason, we have made the mice described here available in a public repository. Although mdx4Cv/NSG and Rag2-Il2rb-Dmd mice were reported 32,33, they are not available in a public repository for easy access by the muscle community. Furthermore, the novel mdx4Cv/NRG mouse reported here may offer additional advantages for the study of muscle diseases. Because NRG mice are more resistant to irradiation, more flexibility in dose of irradiation can be used to eliminate satellite cells, without killing the mice. Less radioresistant mice can also be used for this purpose, using lower doses of irradiation to deplete host regenerative potential. This pre-conditioning creates space in the satellite cell niche for the enhanced engraftment of donor cells with regenerative capacity, assisting the evaluation of candidate therapeutic cells 29. The mdx4Cv/NRG mice reported here may thus be valuable for study of regenerative therapies for DMD. For LGMD2B, Scid/blAJ mice are available [Jackson Labs], but not more immune-deficient strains. For Sgca-/-, there is a lack of immune-deficient mice in public repositories. We have deposited the BlAJ/NRG strain described here to the Jackson Laboratories (Stock No: 029663, BlAJ/NRG, NOD.Cg-Rag1tm1MomDysfprmdIl2rγtmWjl/McalJ), as well as the mdx4Cv/NRG (Stock No: 030442, NOD.Cg-Rag1tm1Mom Dmdmdx-4Cv Il2rγtm1Wjl/McalJ) and Sgca/NRG (Stock No: 030443, NOD.Cg-Rag1tm1Mom Sgcatm1Kcam Il2rγtm1Wjl/McalJ) strains.

The severely immune-deficient mouse models for DMD, LGMD2B, and LGMD2D presented here will find use as key resources to test a variety of cell and gene therapies for these disorders. We validated that each of the mouse models carried the appropriate mutant alleles for the disease gene and immune deficiencies (Figs. 1-3). We also verified that each of the mouse strains was null for the disease gene of interest, using immunohistochemistry and western blots to show the absence of the relevant gene product (Figs. 1-3). To date, we have used the BlAJ/NRG mice in gene therapy experiments and have observed long-term retention (>3 months) of a luciferase transgene, which is otherwise rapidly rejected in immune-competent mice (J. Ma, CP, MB, HdB, and MPC, manuscript in preparation).

We performed characterization of the three mouse models to define their muscle phenotypes, using H&E staining and quantitation of the frequency of centronucleation as a measure of fiber turnover, Evan’s blue staining as a measure of sarcolemmal membrane integrity, and hydroxyproline quantitation as a measure of fibrosis. We also performed a treadmill assay on the mice to measure their ability to perform sustained running. These assays were carried out on mice of a similar age (~6 months), so that we could directly compare the level of pathology present in these three forms of muscular dystrophy disease model mice, although we note that because the lines are not completely inbred, there could be modifier genes that also impact the phenotypes observed. Consistent with the severe phenotypes associated with absence of dystrophin and sarcoglycans, significantly elevated values for the percent of centronucleated fibers were found in the mdx4Cv/NRG and Sgca/NRG mouse models (Fig. 4). These values likely reflect the degeneration and regeneration processes occurring in hind limb muscles as a result of the perturbation of the dystrophin-glycoprotein complex, in which both dystrophin and Sgca participate 1,2,3. Levels of centronucleation were also elevated in the BlAJ/NRG model, but to a lesser degree, consistent with the milder phenotype of dysferlin deficiency.

Evan’s blue dye penetration in six-month old mice was significantly elevated in all muscle groups tested in the mdx4Cv/NRG and Sgca/NRG mice, whereas the effect was predominant only in the hamstring muscle of BlAJ/NRG mice (Fig. 5a). Over time, in BlAJ/NRG mice 15 months old, Evan’s blue dye penetration became significantly elevated in all muscle groups tested (Fig. 6a). This finding is consistent with the progressive nature of muscular dystrophy and earlier involvement of upper limb muscles, which has been visualized previously in a LGMD2B model by luciferase live imaging 4. The collagen accumulation that accompanies the fibrosis process was detected in the gastrocnemius and hamstring muscles of mdx4Cv/NRG and Sgca/NRG animals by six months (Fig. 5b), but took longer to develop in BlAJ/NRG animals. These mice showed significant elevations of hydroxyproline in the upper limb muscles, quadriceps and hamstring, by 15 months of age (Fig. 6b).

NRG control mice were able to run for about 35 minutes under our assay conditions. We detected a significant deficiency in muscle endurance in the BlAJ/NRG mice, with average run time of about half this time. The mdx4Cv/NRG and Sgca/NRG mice were more severely affected, running for <5 minutes under these conditions. Again, the more severe phenotype was similar for both mdx4Cv/NRG and Sgca/NRG and significantly stronger than the effects in the BlAJ/NRG model (Fig. 5c). These characterizations provide useful baseline values that can be used to guide studies of therapeutic interventions when these mouse strains are utilized in preclinical studies of treatments that may be beneficial for these forms of muscular dystrophy.

Acknowledgements

This work was supported by grants to MPC from the California Institute for Regenerative Medicine, the Jain Foundation, the Muscular Dystrophy Association, and the James Kanagy Fund. We thank the Jain Foundation for making BlAJ mice available to us, and Kevin Campbell for providing Sgca-/- mice.

Competing Interests

The authors have declared that no competing interests exist.

Corresponding Author

M.P. Calos, Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, Tel. 650-723-5558; email calos@stanford.edu