The mdx/mTRKO mouse model combines dystrophin-deficiency with telomere dysfunction/shortening, and may be a superior Duchenne muscular dystrophy model as it better recapitulates several of the human characteristics of DMD myopathology (progressive muscle weakness and damage, skeletal muscle fibrosis, diminished muscle stem cell regenerative capacity, dilated cardiomyopathy, heart failure and shortened life-span).
Helen M Blau, Stanford University School of Medicine
Duchenne muscular dystrophy (DMD) is a progressive muscular disorder caused by an imbalance between muscle degeneration and regeneration resulting in muscle degeneration, necrosis, accumulation of fat and fibrosis, and insufficient regeneration/loss of myofibers. The genetic cause of DMD are mutations of the dystrophin muscular dystrophy gene (DMD) on the X chromosome. Both the Dmdmdx (termination codon in exon 23) and Dmdmdx-4Cv (nonsense point mutation in exon 53) mutations in mice are predicted to express a truncated protein. Females heterozygous for either mutation are viable and fertile with no gross phenotypic abnormalities. Homozygous females and hemizygous males are viable and fertile with myopathic features of DMD; although the myopathology is both less severe than the human disease course and variable by mouse strain genetic background.
Specifically, the muscle pathology observed for C57BL/10ScSn-Dmdmdx mice (C57BL/10.mdx ; Stock No. 001801) and B6Ros.Cg-Dmdmdx-4Cv mice (B6.mdx-4Cv ; Stock No. 002378) includes active fiber necrosis, cellular infiltration, a wide range of fiber sizes, and numerous centrally nucleated regenerating fibers. However, despite the absence of dystrophin in skeletal and cardiac muscles, adult C57BL/10.mdx and B6.mdx-4Cv mice fail to exhibit several of features of DMD, including severe muscle weakness, progressive cardiomyopathy and shortened lifespan. In addition, these animals do not show other skeletal muscle characteristics of DMD (such as smaller number of myofibers, accumulation of fat and fibrosis, insufficient myofiber regeneration, and loss of muscle weight).
Differences between the two mutations exist. The Dmdmdx-4Cv allele has very low frequency of reversion to the wildtype allele in skeletal muscle (~10-fold lower frequency than the Dmdmdx mutation). In addition, the Dmdmdx-4Cv mutation affects more of the transcripts arising from alternative promoter usage within the dystrophin gene compared to the Dmdmdx mutation.
While telomere shortening is normally observed over time in mitotically active tissues, muscle tissue exhibits a lower proliferation rate and less telomere shortening with age. However, increased telomere shortening is associated with dystrophic human muscle cells and DMD patients. C57BL/6J mice homozygous for the telomerase RNA component null allele (C57BL/6J.mTR-/- ; Stock No. 004132) lack telomerase activity. Early generation homozygous mice have intact telomeres and appear grossly unaffected and healthy. However, telomere length is progressively shortened with successive generations of breeding mTR-/- mice together; resulting in dysfunction of the reproductive and hematopoietic systems, but little or no skeletal muscle abnormalities.
To investigate how telomere dysfunction affects the severity of muscular dystrophy seen in dystrophin-deficient mice, Dr. Helen M. Blau (Stanford University School of Medicine) created the double mutant mdx/mTR colony (Stock No. 023535) by breeding C57BL/6J-congenic mTR+/- mice with C57BL/6J-congenic Dmdmdx-4Cv mice. Males homozygous for the mTR null allele and hemizygous for the X-linked Dmdmdx-4Cv allele (mTR-/-;Dmdmdx-4Cv/Y), and females homozygous for both alleles (mTR-/-;Dmdmdx-4Cv/mdx-4Cv), are referred to as mdx/mTRKO. When compared to C57BL/10.mdx and B6.mdx-4Cv animals, the mdx/mTRKO mice exhibit additional features of severe human muscular dystrophy: including profound loss of muscle force, poor performance on a treadmill, increased serum creatine kinase levels, accumulation of fibrosis and calcium deposits within skeletal muscle tissues, kyphosis, dilated cardiomyopathy (ventricular dilation), cardiac contractile and conductance dysfunction, heart failure and shortened life-span. The severity of muscle wasting is concomitant with a decline in muscle stem cell regenerative capacity. mdx/mTRKO mice also exhibit telomere erosion (in cardiomyocytes, but not in other heart muscle cells), mitochondrial fragmentation and increased oxidative stress. The dystrophy phenotype becomes more severe with each successive generation of breeding mdx/mTRKO mice together (because such breeding results in progressively shorter telomere lengths with each generation). Specifically, mdx/mTRKO mice bred together for one generation (G1) exhibit cardiac dysfunction by ~80 weeks of age with death first occurring at ~30 weeks of age (~50% survival at 120 weeks of age). mdx/mTRKO G2 mice exhibit cardiac dysfunction by ~32 weeks of age with death first occurring at ~19 weeks of age (~50% survival at 80 weeks of age). mdx/mTRKO G3 mice exhibit cardiac dysfunction by ~8 weeks of age.
These mdx/mTR mice harbor two mutations; the mTR null allele (Terctm1Rdp) and the X-linked muscular dystrophy mutation (Dmdmdx-4Cv).
The mTR null allele was designed by Dr. Ronald DePinho (Albert Einstein College of Medicine) with a neomycin cassette replacing the entire telomerase RNA component gene on chromosome 3. C57BL/6J-congenic mice harboring the mTR null allele are described and available from The Jackson Laboratory Repository as Stock No. 004132. Dr. Helen M. Blau (Stanford University School of Medicine) obtained heterozygous mTR mutant mice on a C57BL/6 genetic background from Dr. DePinho, and then bred them with C57BL/6J wildtype mice for six more generations. The resulting "C57BL6 mTRHet" mice (B6J.mTRHet) were used as described below.
The Dmdmdx-4Cv mutation was created in the laboratory of Dr. Verne M. Chapman (Roswell Park Memorial Institute) by N-ethyl-N-nitrosourea (ENU) treatment. Dmdmdx-4Cv has as a C-to-T transition (resulting in a termination codon) at position 7916 within exon 53 of the dystrophin muscular dystrophy gene (Dmd) on the X chromosome. B6Ros.Cg-Dmdmdx-4Cv mice are described and available from The Jackson Laboratory Repository as Stock No. 002378. Dr. Helen M. Blau (Stanford University School of Medicine) obtained Dmdmdx-4Cv mice on a C57BL/6 genetic background from Jeffrey S. Chamberlain (University of Washington), and then bred them with C57BL/6J wildtype mice for six generations. The resulting "C57BL6 mdx4cv" mice (B6J.mdx4cv) were used as described below.
To generate the double mutant line, Dr. Blau bred B6J.mTRHet mice with B6J.mdx4cv mice. Males heterozygous for the mTR null allele and hemizygous for the mdx4cv allele (called mdx4cv/mTRHet or mdx/mTRHet) were sent to The Jackson Laboratory Repository in 2013. Upon arrival, males were used to cryopreserve sperm. To establish our living mdx/mTR mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664). See the "Health & Care" section for more details on strain maintenance.
|Allele Name||X linked muscular dystrophy 4, Verne Chapman|
|Allele Type||Chemically induced (ENU)|
|Allele Synonym(s)||mdx4cv; mdx4cv; mdx4cv; mdxCv4|
|Gene Symbol and Name||Dmd, dystrophin, muscular dystrophy|
|Gene Synonym(s)||BMD; CMD3B; DXS142; DXS164; DXS206; DXS230; DXS239; DXS268; DXS269; DXS270; DXS272; Dp427; Dp71; Duchenne muscular dystrophy; MRX85; X-linked muscular dystrophy; X-linked muscular dystrophy; dys; mdx; mdx; pke; pke; pyruvate kinase expression|
|Strain of Origin||C3Ha.Cg-Hprt Pgk1|
|Molecular Note||A C to T transition in exon 53 at position 7916 creates a premature stop codon.|
|Mutations Made By|| |
Dr. Verne Chapman (deceased), Roswell Park Memorial Institute
|Allele Name||targeted mutation 1, Ronald DePinho|
|Allele Type||Targeted (Null/Knockout)|
|Allele Synonym(s)||TR-; Terc-; mTR-; mTerc-|
|Gene Symbol and Name||Terc, telomerase RNA component|
|Gene Synonym(s)||mTER; mTR|
|Strain of Origin||STOCK 129/Sv and C57BL/6J and SJL|
|Molecular Note||Replacement of the entire gene with a neomycin cassette.|
|Mutations Made By|| |
Dr. Carol Greider, Johns Hopkins Univ School of Medicine
The Dmdmdx-4Cv mutation is X-linked; therefore mdx-deficient mice are Dmdmdx-4Cv/mdx-4Cv females and Dmdmdx-4Cv/Y males. Telomere length is progressively shortened with successive generations of breeding homozygous mTR null mice (mTR-/-) together.
To replicate the findings in Mourkioti et al. 2013 Nat Cell Biol 15:895, Dr. Helen M. Blau suggests following their specific breeding scheme in Supplementary Fig. S1a of that publication. This scheme places emphasis on starting with the parental generation for every cohort of G0, G1, G2, etc., and using non-sibling matings only as a means of reducing the likelihood of spontaneous mutations influencing experimental outcome. Investigators wishing to recapitulate Dr. Blau's exact breeding scheme may wish to contract with JAX Breeding Services.
The Jackson Laboratory Repository colony began by using Dmdmdx-4Cv/Y;mTR+/- males (G0 males) from Dr. Blau to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664). Next, mice were bred together that were heterozygous or homozygous for Dmdmdx-4Cv and wildtype or heterozygous (not homozygous) for mTR- to obtain animals with G0 genotype. Thereafter, for routine maintenance of the living mdx/mTR colony at The Jackson Laboratory Repository (Stock No. 023535), we bred non-sibling G0 mice together. By this method, the animals with the genotypes listed in part A (i-vi) may be available from The Jackson Laboratory Repository:
A) G0 animals are dystrophin-deficient and heterozygous for mTR. G0 animals have the same phenotype as C57BL/6J-congenic Dmdmdx-4Cv homozygous mice (Stock No. 002378). Breeding G0 animals together (Dmdmdx-4Cv/mdx-4Cv;mTR+/- females and Dmdmdx-4Cv/Y;mTR+/- males) results in the following offspring:
i) Dmdmdx-4Cv/mdx-4Cv;mTR+/+ females
ii) Dmdmdx-4Cv/Y;mTR+/+ males
iii) Dmdmdx-4Cv/mdx-4Cv;mTR+/- females (G0 females)
iv) Dmdmdx-4Cv/Y;mTR+/- males (G0 males)
v) Dmdmdx-4Cv/mdx-4Cv;mTR-/- females (G1 females)
vi) Dmdmdx-4Cv/Y;mTR-/- males (G1 males)
B) G1 animals are the first generation of mice deficient in both dystrophin and mTR. G1 animals have enhanced DMD myopathology. Breeding G1 animals together (Dmdmdx-4Cv/mdx-4Cv;mTR-/- females and Dmdmdx-4Cv/Y;mTR-/- males) results in the following offspring:
i) Dmdmdx-4Cv/mdx-4Cv;mTR-/- females (G2 females)
ii) Dmdmdx-4Cv/Y;mTR-/- males (G2 males)
C) G2 animals are the second generation of mice deficient in both mdx and mTR. G2 animals have enhanced DMD myopathology that is more severe than G1 animals. Breeding G2 animals together results in G3 offspring.
Note that G2, G3, etc. animals may be obtained by contract with JAX Breeding Services.
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