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Human Molecular Genetics Pages 1149-1153
Differential expression of dystrophin isoforms in strains of mdx mice with different mutations
Introduction
Results
Discussion
Materials And Methods
   Mouse strains
   Immunoblot analysis
   Polymerase chain reaction (PCR)
   DNA sequencing
Acknowledgements
References

Differential expression of dystrophin isoforms in strains of mdx mice with different mutations

Differential expression of dystrophin isoforms in strains of mdx mice with different mutations Wha Bin Im1,+, Stephanie F. Phelps2,+, Eecole H. Copen2, Earl G. Adams1, Jerry L. Slightom1 and Jeffrey S. Chamberlain2,*

1CNS Research, The Upjohn Company, 301 Henrietta St., Kalamazoo, MI 49001, USA and 2The Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109-0618, USA

Received April 8, 1996; Revised and Accepted May 24, 1996

Mutations in the dystrophin gene are responsible for Duchenne and Becker muscular dystrophy (DMD/BMD). Studies of dystrophin expression and function have benefited from use of the mdx mouse, an animal model for DMD/BMD. Here we characterized mutations in three additional strains of mdx mice, the mdx2cv, mdx4cv and mdx5cv alleles. The mutation in the mdx2cv mouse was found to be a single base change in the splice acceptor sequence of dystrophin intron 42. This mutation leads to a complex pattern of aberrant splicing that generates multiple transcripts, none of which preserve the normal open reading frame. In the mdx5cv allele, the dystrophin mRNA contains a 53 bp deletion of sequences from exon 10. Analysis of the genomic DNA uncovered a single A to T transversion in exon 10. Although this base change does not alter the encoded amino acid, a new splice donor was created (GTGAG) that generates a frameshifting deletion in the processed mRNA. In the mdx4cv allele, direct sequencing revealed a C to T transition in exon 53, creating an ochre codon (CAA to TAA). The differential location of these mutations relative to the seven known dystrophin promoters results in a series of mdx mouse mutants that differ in their repertoire of isoform expression, such that these mice should be useful for studies of dystrophin expression and function. The mdx4cv and mdx5cv strains may be of additional use in gene transfer studies due to their low frequency of mutation reversion.

INTRODUCTION

Duchenne and Becker muscular dystrophy are human degenerative muscle diseases arising from mutations in the dystrophin gene on the X-chromosome (1 ,2 ). This gene spans approximately 2.4 Mb of DNA and contains at least seven promoters coupled to as many as 79 exons (2 -7 ). As a result, multiple isoforms of dystrophin are expressed in different tissues and at separate stages of development (2 -10 ). Analysis of dystrophin gene expression and function has been aided by studies in mice with dystrophin gene mutations (mdx), of which there are five known alleles (11 ,12 ). Different strains of mdx mice have been reported to display a wide range of reversion frequencies as evidenced by the presence of dystrophin expressing muscle fibers on an otherwise dystrophin deficient background (13 ). Understanding of dystrophin and its isoforms at the molecular level may aid in the development of therapeutic means to ameliorate the symptoms of muscular dystrophy. Most dystrophin studies to date have been carried out with the original mdx mutant, which contains a premature stop codon in exon 23 (11 ,14 ). Four newer strains of mdx mice have been described, mdx2cv-5cv, that were generated with N-ethylnitrosourea (ENU) chemical mutagenesis (12 ). The mdx3cv allele arises from a mutant splice acceptor site in intron 65 (15 ), but the nature of the other mutants has not been characterized.

In this study we investigated the mutation sites in the dystrophin gene of the mdx2cv, mdx4cv and mdx5cv alleles. Together with known mutation data from mdx and mdx3cv mice, studies of dystrophin isoforms can now be performed with mdx mutants that display a wide range of differential expression of the separate dystrophin isoforms.

RESULTS

To identify the genetic lesions in the mdx2cv, mdx4cv and mdx5cv mice, we began scanning the dystrophin gene for abnormalities. Initially we performed western blot analysis of mouse muscle and brain protein extracts using antisera raised against the C-terminal 315 amino acids of murine dystrophin. The results confirmed that none of the five mdx mutants expressed full-length 426 kDa dystrophin in muscle or brain, although very low levels of a 415 kDa mutant dystrophin were observed in mdx3cv muscle (13 ,15 , and data not shown). In contrast, only the mdx3cv animals failed to express the 71 kDa Dp71 isoforms of dystrophin normally found in brain and many other non-muscle tissues (Fig. 1 ). Dp71 expression in the other four mdx strains was not detectably different from that in control C57BL/10 mice, displaying the characteristic pattern of multiple bands resulting from differential phosphorylation and alternative splicing of this isoform (16 ). These results indicated that the mdx2cv, mdx4cv and mdx5cv mutations were located 5' of the beginning of exon 63 (base 9585), into which the Dp71 first exon splices (15 ).


Figure 1.Immunoblot analysis of dystrophin in mdx mouse strains. Protein extracts were prepared from quadriceps muscle (M), brain (B) and liver (L) of the indicated mdx or control C57BL/10 (C57) mouse strains. The proteins were separated on 10% polyacrylamide gels, transferred to nitrocellulose membranes (Bio-trace, Gelman) and analyzed with antisera raised against the C-terminal 315 amino acids of mouse dystrophin (16). The ~71 kDa (Dp71) isoform was observed in brain (and liver) extract from all animals except the mdx3cv mouse.

The remainder of the dystrophin mRNA was examined in a 3' to 5' direction starting at base 9585 by polymerase chain reaction (PCR) amplification of reverse transcribed (RT) RNA isolated from mouse quadriceps muscle. Amplified fragments between 0.5 and 1 kb in size were directly sequenced and compared with the wild-type mouse dystrophin cDNA sequence (17 ). No mutations were identified between bases 9585 and 6977 with this method. However, amplification of the region including bases 6300 to 7000 using multiple primer pairs revealed a complex pattern of splicing in the mdx2cv mouse but not other strains of mice (data not shown). Direct sequencing of these PCR products revealed that a shared feature of each aberrant transcript was the absence of bases 6344 to 6375 from the beginning of exon 43, suggesting a likely mutation in the splice acceptor sequence immediately 5' of exon 43. Inverse PCR was therefore used to amplify the 3' end of intron 42, which upon sequencing revealed an A to T transversion two bases 5' of exon 43. Figure 2 a shows the sequence of the intron 42/exon 43 junction and the site of the mutation. Allele-specific oligonucleotide (ASO) hybridization of the intron 42-exon 43 region showed that an ASO for the mutant sequence hybridized with mdx2cv genomic DNA, while an ASO for the wild-type sequence hybridized with mdx3cv and C57BL/10 mouse DNA, confirming the mutation in the mdx2cv mouse (Fig. 2 b). Sequence analysis of a number of aberrant splicing products isolated from mdx2cv muscle RNA revealed that exon 42 sequences were spliced into the middle of exon 43 (Fig. 2 a), which generally spliced normally to exon 44. However, 3' of exon 44 a variety of splicing events were identified that retained various combinations of exons 45 through 51. None of these splice forms preserved the normal open reading frame, explaining the absence of detectable dystrophin in mdx2cv muscle (Fig. 1 ).


Figure 2. (A) Sequence of the mouse dystrophin gene intron 42/exon 43 junction. Inverse PCR was used to isolate the 3' end of intron 42 (see methods), generating the sequence shown. The A to T transversion is indicated at the 3' end of intron 42. The base designated by +1 represents the site into which aberrantly spliced transcripts were found to be joined to sequences from the 3' end of exon 42 in mdx2cv muscle RNA. The cryptic splice acceptor sequence is underlined. An in-frame stop codon (boxed) is in the reading frame of those transcripts. (B) ASO hybridization of amplified genomic DNA from the dystrophin intron 42/exon 43 junction. Primers 2072D and 2071D (see A) were used to amplify genomic DNA from the indicated strains of mice, which was slot-blotted onto a nylon membrane, and hybridized with an ASO specific for the wild-type sequence or the mdx2cv sequence. The dotted line below the sequence indicates the region used for hybridization probes. The results indicate that the mdx2cv sequence alteration is not present in DNA from C57BL/10 or mdx3cv mice [the latter of which has the same genetic background as mdx2cv mice (14)].

To identify the mutations in the mdx4cv and mdx5cv mice we analyzed RT-PCR products generated from quadriceps muscle RNA spanning bases 128 to 9585 of the murine dystrophin cDNA sequence, each primer pair amplifying on average a region of about 800 bp (see Methods). From agarose gel electrophoresis, all the PCR products appeared to be of the expected size except for one obtained with the primer set covering the region between bases 752 and 1712. The PCR products from wild-type C57Bl/10 mice and mdx4cv mice had the expected size of 961 bp, but those from the mdx5cv mouse were noticeably shorter (908 bp, see below). Direct sequencing of the mdx5cv PCR product, using a primer that anneals with the region between bases 1487 and 1464 of the cDNA, revealed a deletion of 53 bp that extended from the middle to the very 3' end of exon 10 (bases 1323 to 1375). This deletion of exon 10 sequences shifted the mRNA translational reading frame, which now contained an ochre (UAA) termination codon at base 1416 (numbering refers to the wild-type sequence) (17 ).

To find the mutation responsible for this mRNA deletion, genomic DNA from mdx5cv mice was amplified with a forward primer corresponding to the region between bases 1193 and 1212 (at the beginning of exon 10) and a reverse primer targeted to bases 1374-1354 (at the end of exon 10). Agarose gel electrophoresis indicated that the genomic PCR product was of the expected size (182 bp). Direct sequencing, however, revealed an A to T transversion at the position equivalent to dystrophin cDNA base 1324 (17 ). This mutation produced a consensus splice donor site (GTGAG), accounting for the aberrant splicing of exon 10 sequences (Fig. 3 ).


Figure 3.Sequence analysis of exon 10 from genomic DNA of the mdx5cv mouse. Direct sequencing of the PCR product, obtained using a primer set covering the beginning and end of exon 10, showed an A to T mutation at base 1324. This mutation created a new splice donor site in the middle of exon 10 (underlined). The dystrophin mRNA in the mdx5cv mice splices in such a way as to join base 1322 to the first base of exon 11 (base 1376).

Agarose gel electrophoresis indicated that all the PCR products from the mdx4cv cDNA were of the expected size. However, genetic mapping had suggested the mdx4cv mutation was located in the 3' half of the gene (15 ), so PCR products from base 7000 to 9585 were directly sequenced. This analysis uncovered a mutation at base 7916, changing a C to a T, producing a premature stop codon (ochre codon, TAA; Fig. 4 ). No other mutations were detected from sequencing the products covering other regions of the dystrophin mRNA.


Figure 4.Sequence analysis of the region between bases 7892 and 7930 of the dystrophin cDNA from mdx4cv mice in comparison with the wild type (C57) sequence. Direct sequencing of the PCR products revealed a C to T mutation at the base 7916, creating a nonsense codon.

DISCUSSION

In this study we characterized mutations in the mdx2cv, mdx4cv and mdx5cv strains of mice. All three strains were found to have point mutations, as has also been previously found in the mdx and the mdx3cv mouse strains (14 ,15 ). In contrast, 60-70% of mutations in the human dystrophin gene are deletions or duplications (reviewed in 18 ). Of the five mouse mutations currently known, only the original mdx allele is a naturally occurring lesion. The four other alleles arose from ENU mutagenesis, which typically generates point mutations (12 ). Thus the question of whether the mouse dystrophin gene is also prone to deletions can not be directly answered from these results. Of the five mdx strains, only the mdx3cv mice accumulate detectable dystrophin in muscle (13 ,15 ). However, this latter mutant protein is apparently non-functional (19 ), and all five mdx strains display an essentially identical muscle pathology (12 ,13 ,15 ). A useful feature of the separate alleles is the location of the mutations relative to the dystrophin gene promoters (Fig. 5 ). Studies utilizing the ENU-derived mutants may reveal subtle pathological abnormalities in non-muscle tissues, and may aid in defining the precise expression patterns of the C-terminal dystrophin isoforms.

Of the five mutations in the mouse dystrophin gene, two are nonsense mutations while three affect mRNA splicing. In the mdx5cv mice, analysis of genomic DNA uncovered an A to T mutation in the middle of exon 10 that produced a new splice donor site. RT-PCR analysis of muscle RNA from the mdx5cv mouse indicated that this cryptic splice site is exclusively used (data not shown). Interestingly, this mutation would not have been considered responsible for the phenotype if our analysis had been limited to genomic DNA sequencing, as the base change does not alter the amino acid specified by the codon (GGA to GGT, both encode glycine). Analysis of the mRNA was needed to reveal the presence of a cryptic splice site, which generates a premature stop codon in the full-length transcripts. The location of this mutation indicates that the mdx5cv mouse should display an identical pattern of dystrophin isoform expression as the original mdx mouse, which has a nonsense mutation in exon 23 (14 ; Fig. 5 ).

The mdx2cv allele also results from a mutation affecting mRNA splicing, and is located in the splice acceptor of intron 42. Previously we demonstrated that the mdx3cv mutation is in the splice acceptor of intron 65 (15 ). However, in the case of the mdx3cv allele, alternative splicing of the resultant transcript leads to low level accumulation of a dystrophin protein lacking cysteine rich domain sequences encoded by exons 65 and 66 (15 ). We have shown that this internally truncated protein is non-functional when expressed in transgenic mdx mice, explaining why the mdx3cv muscle phenotype is indistinguishable from that of the other mdx mice (19 ). Similar to the mdx3cv allele, the mdx2cv splice acceptor mutation generates a complex pattern of alternatively spliced transcripts. However, in contrast to the mdx3cv mutation, we were unable to find an alternative transcript preserving the normal open reading frame. The location of the mdx2cv mutation predicts that transcripts originating from the retinal (R) promoter will be defective, in addition to those arising from the 5' gene promoters active in muscle (M), and brain cortical (C) and Purkinje cell (P) regions (1 ,3 ,6 ,10 ; see Fig. 5 ).

In the case of the mdx4cv allele, direct sequencing revealed a C to T transition at base 7916 which changes the codon CAA to TAA, a nonsense mutation (a premature termination of polypeptide synthesis). In an earlier linkage study, restriction fragment length polymorphism analysis with human dystrophin cDNA probe 8 (1 ) had mapped the mdx4cv mutation to a position 3' from murine cDNA base 7014 (15 ). This prediction is in agreement with our current finding of the C to T mutation at base 7916. The chain terminating mutation in the mdx4cv strain is considerably downstream from the retinal promoter and from the `brain' promoter located in intron 44 (6 ,7 ). The mdx4cv strain may therefore prove useful in studies of the function of non-muscle dystrophin isoforms expressed from internal promoters (Fig. 5 ).


Figure 5.Location of the five mouse mdx mutations, relative to the seven dystrophin promoters and the shared exon into which each unique first exon splices. Vertical arrows corresponding to the site of each mdx mutation intersect horizontal bars representing each of the five size classes of transcripts that arise from the seven promoters. To the right of the gene diagram is indicated which isoforms of dystrophin are expressed in each of the five strains of mdx mice.

The mutation in the mdx5cv mouse is considerably upstream from the original mdx mutation (a C to T mutation at base 3203), and the mutated murine dystrophin is expected to be the shortest known so far. Both the mdx5cv and mdx4cv mice display a low background of reverent fibers, about 10 times fewer than the mdx or mdx2cv alleles (13 ). This low reversion rate suggests that these mice might serve as useful strains for studies needing a low background of dystrophin-containing fibers, such as gene transfer studies exploring the feasibility of DMD therapy. The relative location of the mutations in the mdx strains should also facilitate analysis of the role of the multiple dystrophin isoforms.

MATERIALS AND METHODS

Mouse strains

The mdx2-5cv congenic mouse strains were generously provided by the late Dr Verne Chapman, and are currently being propagated at The Jackson Laboratories (Bar Harbor, ME). Control C57BL/10 and mutant C57BL/10mdx mice were bred at the University of Michigan from stocks originally obtained from The Jackson Laboratories.

Immunoblot analysis

Preparation of protein extracts, dystrophin antisera, and immunoblotting were performed as described previously (15 ,16 ,19 ,20 ).

Polymerase chain reaction (PCR)

Overlapping primers for PCR were designed to amplify sequences between base 128 to base 9585 of the wild-type C57BL/10 mouse dystrophin cDNA [C57M; (17 )]. The length of expected PCR products ranges from 400 to 1000 bp with an average size of about 800 bp. The following lists the overlapping primer sets which were utilized. In the parentheses, the location of the forward primer was indicated with the two base numbers of the dystrophin cDNA denoting the beginning and end of the primer, followed by the second set of base numbers for the reverse primer: (128-151:1080-1057); (752-776:1712-1690); (1655- 1676:2621-2599); (2343-2365:3166-3143); (3064-3087: 3963- 3940); (3733-3755:4637-4616); (4532-4553:5530-5509); (5432- 5453:6200-6176); (5635-6416: 6416-6394); (6313-6334: 7153- 7132); (6977-7001:7332-7316); (7131-7149:7645- 7621); (7253-7547:8164-8146); (8051-8068:8653-8638); (8330-8352: 9228- 9205) and (9090-9113:9585-9563). PCRs were performed in a 50 [mu]l volume using 1 U Amplitaq DNA polymerase (Perkin-Elmer Cetus) in the vender supplied buffer, 200 [mu]M each dNTP, 1 [mu]g of cDNA obtained by reverse transcription of quadriceps muscle total RNA, and 10 pmol of each primer. The cycle parameters were at 94oC for 30 s, 54oC for 30 s (variable from 52-56oC depending on the primer pair), and 72oC for 60 s, with a final extension at 72oC for 10 min after 35 cycles.

Genomic DNA was obtained from mdx5cv liver and used as a template for PCR with a forward primer spanning bases 1193 to 1212 of the dystrophin cDNA (at the beginning of exon 10) and a reverse primer spanning bases 1374 to 1354 (at the end of exon 10). Inverse PCR to isolate intron 42/exon 43 sequences was performed as described previously (15 ), except that primers 9876C (Fig. 2 a) and 9877C (bases 6497-6516) were used to amplify restriction digested and recircularized liver genomic DNA from C57BL/10 and mdx2cv mice prior to direct sequence analysis. PCR for ASO hybridizations used primers 2072D and 9876C (Fig. 2 a), and 150 ng genomic DNA in a 25 [mu]l reaction (40 cycles at 50oC, 72oC, and 94oC, 15 s each). Twenty-five ng of this product was diluted to 0.1 M NaCl, slot-blotted onto Nytran membranes (Schleicher and Schuell), and hybridized as described (21 ) using end-labeled oligonucleotide Int42-wt (5'-TTA-TTT-CAG-AAT-ATA-3'), which hybridizes with the wild-type intron 42-exon 43 junction, and oligonucleotide Int42-2cv (5'-TTA-TTT-CTG-AAT-ATA-3'), which hybridizes with the mdx2cv intron 42-exon 43 junction. Blots were washed as described (21 ) for 10 min at 22oC.

DNA sequencing

Direct sequencing was carried out using a PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing Kit from Applied Biosystems, Inc. Sequencing reaction mixtures contained a PCR product of interest (10 ng), AmpliTaq, a mixture of four DyeDeoxy terminators, and a primer (0.8 pmol in 1 [mu]l) in the vendor supplied buffer (20 [mu]l). The extension/amplification reactions were carried out using an automated PCR thermal cycler (Perkin-Elmer GeneAmp 9600), with an initial denaturation at 98oC for 1 min, followed by 35 cycles of 98oC for 14 s, 50oC for 15 s and 60oC for 4 min. The products were purified by spin column (CentriSep Columns, Princeton Separations, Inc), dried under vacuum and dissolved in 4 [mu]l of a DNA loading solution (83% deionized formamide, 8.3 mM EDTA, and 16 mg/ml blue dextran). The denatured samples were resolved in a sequencing gel containing 4.75% polyacrylamide and 7.5 M urea on an ABI 373A DNA sequencer. Both DNA strands were sequenced for the fragments described here.

ACKNOWLEDGMENTS

The authors are indebted to the late Verne Chapman for generation of mdx mutants and for generously supplying the mice used in this study. We thank C. Thomas Caskey, Carey Lumeng, Greg Cox, and Jill Rafael for helpful discussions. Supported by a grant from the Muscular Dystrophy Association (USA), by NIH grant AR40864 (to JSC), and by The Upjohn Company.

REFERENCES

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2 Koenig, M., Hoffman, E. P., Bertelson, C. J., Monaco, A. P., Feener, C. and Kunkel, L. M. (1987) Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell, 50, 509-517. MEDLINE Abstract

3 Nudel, U., Zuk, D., Einet, P., Zeelon, E., Levy, Z., Neuman, S. and Yaffe, D. (1989) Duchenne muscular dystrophy gene product is not identical in muscle and brain. Nature, 337, 76-78. MEDLINE Abstract

4 Bar, S., Barnea, E., Levy, Z., Neuman, S., Yaffe, D. and Nudel, U. (1990) A novel product of the Duchenne muscular dystrophy gene which greatly differs from the known isoforms in its structure and tissue distribution. Biochem. J., 272, 557-560. MEDLINE Abstract

5 Byers, T. J., Lidov, H. G. W. and Kunkel, L. M. (1993) An alternative dystrophin transcript specific to peripheral nerve. Nature Genet, 4, 77-81. MEDLINE Abstract

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7 Lidov, H. G. W., Selig, S. and Kunkel, L. M. (1995) Dp140: A novel 140 kDa CNS transcript from the dystrophin locus. Hum. Mol. Genet., 4, 329-335.

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9 Bies, R. D., Phelps, S. F., Cortez, M. D., Roberts, R., Caskey, C. T. and Chamberlain, J. S. (1992) Human and murine dystrophin mRNA transcripts are differentially expressed during skeletal muscle, heart, and brain development. Nucleic Acids Res., 20, 1725-1731. MEDLINE Abstract

10 Gorecki, D. C., Monaco, A. P., Derry, J. M., Walker, A. P., Barnard, E. A. and Barnard, P. J. (1992) Expression of four alternative dystrophin transcripts in brain regions regulated by different promoters. Hum. Mol. Genet., 1, 505-510. MEDLINE Abstract

11 Bulfield, G., Siller, W. G., Wight, P. A. and Moore, K. J. (1984) X chromosome-linked muscular dystrophy (mdx) in the mouse. Proc. Natl. Acad. Sci. USA, 81, 1189-1192. MEDLINE Abstract

12 Chapman, V. M., Miller, D. M., Armstrong, D. and Caskey, C. T. (1989) Recovery of induced mutations for X chromosome-linked muscular dystrophy in mice. Proc. Natl. Acad. Sci. USA, 86, 1292-1296. MEDLINE Abstract

13 Danko, I., Chapman, V. and Wolff, J. A. (1992) The frequency of revertants in mdx mouse genetic models for Duchenne muscular dystrophy. Pediatr. Res., 32, 128-131. MEDLINE Abstract

14 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. MEDLINE Abstract

15 Cox, G. A., Phelps, S. F., Chapman, V. M. and Chamberlain, J. S. (1993) New mdx mutation disrupts expression of muscle and nonmuscle isoforms of dystrophin. Nature Genet, 4, 87-93. MEDLINE Abstract

16 Cox, G. A., Sunada, Y., Campbell, K. P. and Chamberlain, J. S. (1994) Dp71 can form a stable dystrophin-associated glycoprotein complex in muscle but fails to prevent dystrophy. Nature Genet, 8, 333-338. MEDLINE Abstract

17 Chamberlain, J. S., Pearlman, J. A., Muzny, D. M., Civetello, A., Farwell, N. J., Malek, R., Powaser, P., Reeves, A. A., Lee, C.C. and Caskey, C. T. (Genbank M68859).

18 Amalfitano, A., Rafael, J. A. and Chamberlain, J. S. (1996) Dystrophin gene structure and mutation. In: Lucy J and Brown S, eds., `Dystrophin: Gene, Protein and Cell Biology'. Cambridge University Press, in press.

19 Rafael, J. A., Cox, G. A., Jung, D., Campbell, K. P. and Chamberlain J. S. (1996) Forced expression of dystrophin deletion constructs reveals structure-function correlations. J. Cell Biol., in press. MEDLINE Abstract

20 Bulman, D. E., Murphy, E. G., Zubrzycka-Gaarn, E. E., Worton, R. G. and Ray, P. N. (1991) Differentiation of Duchenne and Becker muscular dystrophy phenotypes with amino- and carboxy-terminal antisera specific for dystrophin. Am. J. Hum. Genet., 48, 295-304. MEDLINE Abstract

21 Chamberlain, J.S. Farwell, N. J., Ranier, J. E., Cox, G. A. and Caskey C. T. (1991) PCR analysis of dystrophin gene mutation and expression. J. Cell Biol., 46, 255-259.

*To whom correspondence should be addressed+The two first authors contributed equally to this work

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