Generation and Characterization of Transgenic Mice with the Full-length Human DMD Gene

Abstract

We report the generation of mice with an intact and functional copy of the 2.3-megabase human dystrophin gene (hDMD), the largest functional stretch of human DNA thus far integrated into a mouse chromosome. Yeast spheroplasts containing an artificial chromosome with the full-length hDMD gene were fused with mouse embryonic stem cells and were subsequently injected into mouse blastocysts to produce transgenic hDMD mice. Human-specific PCR, Southern blotting, and fluorescent in situ hybridization techniques demonstrated the intactness and stable chromosomal integration of the hDMD gene on mouse chromosome 5. Expression of the transgene was confirmed by RT-PCR and Western blotting. The tissue-specific expression pattern of the different DMD transcripts was maintained. However, the human Dp427p and Dp427m transcripts were expressed at 2-fold higher levels and human Dp427c and Dp260 transcripts were expressed at 2- and 4-fold lower levels than their endogenous counterparts. Ultimate functional proof of the hDMD transgene was obtained by crossing of hDMD mice with dystrophin-deficient mdx mice and dystrophin and utrophin-deficient mdx × Utrn-/- mice. The hDMD transgene rescued the lethal dystrophic phenotype of the mdx × Utrn-/- mice. All signs of muscular dystrophy disappeared in the rescued mice, as demonstrated by histological staining of muscle sections and gene expression profiling experiments. Currently, hDMD mice are extensively used for preclinical testing of sequence-specific therapeutics for the treatment of Duchenne muscular dystrophy. In addition, the hDMD mouse can be used to study the influence of the genomic context on deletion and recombination frequencies, genome stability, and gene expression regulation. We report the generation of mice with an intact and functional copy of the 2.3-megabase human dystrophin gene (hDMD), the largest functional stretch of human DNA thus far integrated into a mouse chromosome. Yeast spheroplasts containing an artificial chromosome with the full-length hDMD gene were fused with mouse embryonic stem cells and were subsequently injected into mouse blastocysts to produce transgenic hDMD mice. Human-specific PCR, Southern blotting, and fluorescent in situ hybridization techniques demonstrated the intactness and stable chromosomal integration of the hDMD gene on mouse chromosome 5. Expression of the transgene was confirmed by RT-PCR and Western blotting. The tissue-specific expression pattern of the different DMD transcripts was maintained. However, the human Dp427p and Dp427m transcripts were expressed at 2-fold higher levels and human Dp427c and Dp260 transcripts were expressed at 2- and 4-fold lower levels than their endogenous counterparts. Ultimate functional proof of the hDMD transgene was obtained by crossing of hDMD mice with dystrophin-deficient mdx mice and dystrophin and utrophin-deficient mdx × Utrn-/- mice. The hDMD transgene rescued the lethal dystrophic phenotype of the mdx × Utrn-/- mice. All signs of muscular dystrophy disappeared in the rescued mice, as demonstrated by histological staining of muscle sections and gene expression profiling experiments. Currently, hDMD mice are extensively used for preclinical testing of sequence-specific therapeutics for the treatment of Duchenne muscular dystrophy. In addition, the hDMD mouse can be used to study the influence of the genomic context on deletion and recombination frequencies, genome stability, and gene expression regulation. The dystrophin gene (DMD) is the largest gene identified in the human genome. It is located on the X chromosome (band Xp21.2), spans 2.3 Mb, 2The abbreviations used are:Mbmegabase(s)DMDDuchenne muscular dystrophyAONantisense oligonucleotideYACyeast artificial chromosomeESembryonic stemRTreverse transcriptionFISHfluorescent in situ hybridizationCOBRAcombined binary ratio labelingCKcreatine kinase.2The abbreviations used are:Mbmegabase(s)DMDDuchenne muscular dystrophyAONantisense oligonucleotideYACyeast artificial chromosomeESembryonic stemRTreverse transcriptionFISHfluorescent in situ hybridizationCOBRAcombined binary ratio labelingCKcreatine kinase. and contains 79 exons (1den Dunnen J.T. Bakker E. van Ommen G.J. Pearson P.L. Br. Med. Bull. 1989; 45: 644-658Crossref PubMed Scopus (13) Google Scholar, 2Koenig M. Hoffman E.P. Bertelson C.J. Monaco A.P. Feener C. Kunkel L.M. Cell. 1987; 50: 509-517Abstract Full Text PDF PubMed Scopus (1899) Google Scholar, 3Koenig M. Monaco A.P. Kunkel L.M. Cell. 1988; 53: 219-226Abstract Full Text PDF PubMed Scopus (1252) Google Scholar). Transcripts originating from seven different promoters throughout the gene have been described, whereas, in addition, extensive differential splicing of these transcripts has been acknowledged (details can be found on the Leiden Muscular Dystrophy Web pages). The Dp427m transcript is most prominent in skeletal and cardiac muscle. This transcript gives rise to the production of the 427-kDa muscle isoform of the dystrophin protein (4Hoffman E.P. Brown Jr., R.H. Kunkel L.M. Cell. 1987; 51: 919-928Abstract Full Text PDF PubMed Scopus (3588) Google Scholar). In muscle, dystrophin is localized on the cytoplasmic side of the sarcolemma (5Ervasti J.M. Campbell K.P. Cell. 1991; 66: 1121-1131Abstract Full Text PDF PubMed Scopus (1097) Google Scholar). Together with the dystroglycan transmembrane protein complex, it forms the dystrophin-glycoprotein complex, which links the cytoskeletal actin filaments and the extracellular matrix and is essential for the force transduction in the muscle fiber (6Michele D.E. Campbell K.P. J. Biol. Chem. 2003; 278: 15457-15460Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar). The dystrophin protein consists of an N-terminal domain that interacts with actin, a central rod and cysteine-rich domain, and a C-terminal domain that binds to β-dystroglycan. More recently, dystrophin has been implicated in signal transduction, probably via interactions with syntrophin, calmodulin, and nitric-oxide synthase (7Rando T.A. Muscle Nerve. 2001; 24: 1575-1594Crossref PubMed Scopus (295) Google Scholar). The dystrophin protein is highly conserved in vertebrates. Mouse dystrophin has 91% amino acid identity with its human counterpart. Utrophin is a homologue of dystrophin that is mainly functional at the neuromuscular junction (8Tinsley J.M. Blake D.J. Roche A. Fairbrother U. Riss J. Byth B.C. Knight A.E. Kendrick-Jones J. Suthers G.K. Love D.R. Nature. 1992; 360: 591-593Crossref PubMed Scopus (346) Google Scholar). It can substitute for dystrophin in the dystrophin-glycoprotein complex (9Matsumura K. Ervasti J.M. Ohlendieck K. Kahl S.D. Campbell K.P. Nature. 1992; 360: 588-591Crossref PubMed Scopus (440) Google Scholar, 10Tinsley J. Deconinck N. Fisher R. Kahn D. Phelps S. Gillis J.M. Davies K. Nat. Med. 1998; 4: 1441-1444Crossref PubMed Scopus (468) Google Scholar). megabase(s) Duchenne muscular dystrophy antisense oligonucleotide yeast artificial chromosome embryonic stem reverse transcription fluorescent in situ hybridization combined binary ratio labeling creatine kinase. megabase(s) Duchenne muscular dystrophy antisense oligonucleotide yeast artificial chromosome embryonic stem reverse transcription fluorescent in situ hybridization combined binary ratio labeling creatine kinase. Mutations in the dystrophin gene are responsible for Duchenne/Becker Muscular Dystrophy and X-linked dilated cardiomyopathy (11den Dunnen J.T. Grootscholten P.M. Bakker E. Blonden L.A. Ginjaar H.B. Wapenaar M.C. van Paassen H.M. van Broeckhoven C. Pearson P.L. van Ommen G.J. Am. J. Hum. Genet. 1989; 45: 835-847PubMed Google Scholar). Mutations that disrupt the reading frame of the DMD transcript cause Duchenne muscular dystrophy (DMD), a severe form of muscular dystrophy with an incidence of 1 in 3,500. It is characterized by progressive muscle weakness and premature death from cardiac or respiratory failure. Inframe deletions are responsible for the milder Becker muscular dystrophy. The mdx mouse is a genetically homologous mouse model for DMD with a mutation in exon 23 of the Dmd gene. Although genotypically similar, major phenotypic differences exist between the mdx mouse model and human DMD patients. Mdx mice show very effective muscle regeneration, leading to an almost complete recovery from the muscle wasting in early life (12Coulton G.R. Morgan J.E. Partridge T.A. Sloper J.C. Neuropathol. Appl. Neurobiol. 1988; 14: 53-70Crossref PubMed Google Scholar, 13Turk R. Sterrenburg E. de Meijer E.J. van Ommen G.J. den Dunnen J.T. Hoen't P.A. BMC Genomics. 2005; 6: 98Crossref PubMed Scopus (83) Google Scholar). Crossing of mdx mice with utrophin-deficient mice gives a much more severe phenotype, which is more similar to DMD (14Deconinck A.E. Rafael J.A. Skinner J.A. Brown S.C. Potter A.C. Metzinger L. Watt D.J. Dickson J.G. Tinsley J.M. Davies K.E. Cell. 1997; 90: 717-727Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar, 15Grady R.M. Teng H. Nichol M.C. Cunningham J.C. Wilkinson R.S. Sanes J.R. Cell. 1997; 90: 729-738Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar). These mdx × Utrn-/- mice suffer from extreme muscle weakness and die within 20 weeks. A therapy for DMD is currently not available. Among the most prominent therapeutic approaches that are tested in preclinical and clinical studies is reading frame correction by antisense oligonucleotide (AON)-induced modulation of splicing (16van Deutekom J.C. van Ommen G.J. Nat. Rev. Genet. 2003; 4: 774-783Crossref PubMed Scopus (172) Google Scholar, 17Aartsma-Rus A. Janson A.A. Kaman W.E. Bremmer-Bout M. den Dunnen J.T. Baas F. van Ommen G.J. van Deutekom J.C. Hum. Mol. Genet. 2003; 12: 907-914Crossref PubMed Scopus (217) Google Scholar, 18Lu Q.L. Mann C.J. Lou F. Bou-Gharios G. Morris G.E. Xue S.A. Fletcher S. Partridge T.A. Wilton S.D. Nat. Med. 2003; 9: 1009-1014Crossref PubMed Scopus (327) Google Scholar, 19Lu Q.L. Rabinowitz A. Chen Y.C. Yokota T. Yin H. Alter J. Jadoon A. Bou-Gharios G. Partridge T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 198-203Crossref PubMed Scopus (343) Google Scholar, 20Mann C.J. Honeyman K. Cheng A.J. Ly T. Lloyd F. Fletcher S. Morgan J.E. Partridge T.A. Wilton S.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 42-47Crossref PubMed Google Scholar, 21Alter J. Lou F. Rabinowitz A. Yin H. Rosenfeld J. Wilton S.D. Partridge T.A. Lu Q.L. Nat. Med. 2006; 12: 175-177Crossref PubMed Scopus (429) Google Scholar, 22Goyenvalle A. Vulin A. Fougerousse F. Leturcq F. Kaplan J.C. Garcia L. Danos O. Science. 2004; 306: 1796-1799Crossref PubMed Scopus (400) Google Scholar, 23McClorey G. Moulton H.M. Iversen P.L. Fletcher S. Wilton S.D. Gene Ther. 2006; 13: 1373-1381Crossref PubMed Scopus (172) Google Scholar). Since AONs are sequence-specific, AONs be tested in This was of the generation of the hDMD demonstrated in an report the of hDMD transgenic mice for of the of antisense M. A. de Meijer E.J. Kaman W.E. Janson A.A. R.H. van Ommen G.J. den Dunnen J.T. van Deutekom J.C. Mol. Ther. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). report the of the hDMD transgenic mice and the of the hDMD from its in the preclinical of sequence-specific of mouse model in more of hDMD of a yeast artificial chromosome with the full-length human DMD gene and of embryonic stem from mice from a × with yeast spheroplasts containing the have been Dunnen J.T. Grootscholten P.M. J.G. A.P. Monaco A.P. R. R. A.J. D.R. Hum. Mol. Genet. 1992; PubMed Google Scholar, C. PubMed Google Scholar). were by Southern PCR, in situ hybridization and RT-PCR and Western DMD and stable genomic and expression of the hDMD gene were injected into blastocysts that were subsequently in the mice with levels of were and to of the hDMD was by DNA DMD and transgenic hDMD mice were obtained that were for expression of the human dystrophin gene. The of these mice was by the of and in mice that were with The for were used for for the gene expression hDMD mice from with a more × × were mdx mice were obtained from Utrn-/- and Utrn-/- × mdx mice were (14Deconinck A.E. Rafael J.A. Skinner J.A. Brown S.C. Potter A.C. Metzinger L. Watt D.J. Dickson J.G. Tinsley J.M. Davies K.E. Cell. 1997; 90: 717-727Abstract Full Text Full Text PDF PubMed Scopus (587) Google Scholar). In gene expression profiling mice with a × × were used as to the of the mdx and hDMD mice as as were and were was on of from mice. The is in K. Nat. Scopus Google were by labeling of by with a of fluorescent and K. Nat. Scopus Google Scholar). The of the chromosome used for of the hDMD gene were from and exons and exons was with which was by with by by was with which was by with mouse by and was from mouse by at with of and of for at 1 of the was used for of of of and 1 of with exon and exon reverse The was with of in 1 for at The was with the In the were to a containing of and for 1 to for the were to a containing of and for 1 by and the the were to a containing of and 20 of were for 1 at to and into the of the The was and the were to the and RT-PCR and were as M. A. de Meijer E.J. Kaman W.E. Janson A.A. R.H. van Ommen G.J. den Dunnen J.T. van Deutekom J.C. Mol. Ther. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, J.M. de Meijer E.J. van Ommen den Dunnen J.T. 12: Full Text Full Text PDF PubMed Scopus Google Scholar). were on the as the a was by of a from of the and were used as of the different dystrophin and were in homologous the was in the for the transcript and the reverse in a are in the for differences in between hDMD and were for the differences in gene the was to the expression of the transcript in a the to in the mice, the Western on muscular protein was as A. Janson A.A. Kaman W.E. Bremmer-Bout M. den Dunnen J.T. Baas F. van Ommen G.J. van Deutekom J.C. Hum. Mol. Genet. 2003; 12: 907-914Crossref PubMed Scopus (217) Google Scholar). The and were obtained from G. Morris and and sections from muscle were with a and of these sections were as M. A. de Meijer E.J. Kaman W.E. Janson A.A. R.H. van Ommen G.J. den Dunnen J.T. van Deutekom J.C. Mol. Ther. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). of the dystrophin-glycoprotein complex, with mouse and from a and the were the sections were with the J.T. PubMed Scopus Google with an was by staining of of the complete lower with acid to L. PubMed Google Scholar). In of from the from mice, was in with was by for at in an was by of of on a and in the Gene Expression expression of with the was used to a of was on gene as J.M. de Meijer E.J. van Ommen den Dunnen J.T. 12: Full Text Full Text PDF PubMed Scopus Google Scholar). were into and with the model for of 1 × was used to expressed between dystrophic and Utrn-/- × and mice hDMD × hDMD × Utrn-/- × in gene expression were by the Pearson was used as a were to the Gene Expression and have In were used in the of hDMD of yeast cells a of that contains the full-length human dystrophin gene Dunnen J.T. Grootscholten P.M. J.G. A.P. Monaco A.P. R. R. A.J. D.R. Hum. Mol. Genet. 1992; PubMed Google with cells and of stable copy the cells were injected into These were subsequently in the mice with the levels of were tested for and with mice to and and mice. of and on from with different exons and of the hDMD gene demonstrated copy integration of the hDMD gene into mouse chromosome to the of the highly homologous DMD of the hDMD mice was A was to between and mice. In the signal is from the of of a at a A between the mouse and human isoform in a conserved in exon of the dystrophin gene in of in of a human and and in of a mouse The mice can be on the The were higher than This is probably to the higher of However, the between the and was highly as demonstrated by S.D. and ratio and mice ratio The more than a not and of and hDMD mice are at the have functional and have a and life RT-PCR with in of the from the to the the expression of full-length human dystrophin in the muscle of hDMD mice. of an RT-PCR the of human and mouse dystrophin is in of protein and sections from the muscle of hDMD mice by Western and staining with the demonstrated the of a full-length 427-kDa human dystrophin protein at the sarcolemma and The levels of human and mouse dystrophin protein by staining with the were similar to the levels of the protein in mice. We the tissue-specific expression of transcripts originating from the DMD gene were in mice. of the RT-PCR of the and human and transcripts in the different of hDMD and mice are in of to be All hDMD transcripts were expressed in the as their endogenous the and Dp260 most prominent in the the in the and the in the muscle and in homologous and a of the expression of the human and mouse transcripts in and mice. Although similar, differences in In mice, 2-fold higher expression of the human Dp427p and Dp427m lower expression of the human Dp427c and Dp260 transcripts and with their and In the expression of the endogenous mouse transcript is lower in the than in the mice of a of the human protein on the transcription of the gene. of by hDMD the of the hDMD the hDMD mice with dystrophin-deficient mdx mice. × mdx mice not show signs of muscular dystrophy or Together with the dystrophin the dystrophin-glycoprotein complex was at the as by staining for and In mdx mice of in sections and as by staining with an J.T. PubMed Scopus Google We have not differences in fiber between rescued and mice. are in In the muscle The muscle of mice of 23 were found in the muscle of × mdx mice not with a of The which are in dystrophic mice to of were in × mdx mice The combined that a copy of the hDMD gene the mdx A more is in × mice, which are and die between and 20 In the incidence of premature death in the × Utrn-/- × mdx mice was not different from that in mice, and the rescued mouse in to the of which it was The mice have muscle and not signs of histological of of these mice not show signs of muscular dystrophy were fiber was and was not In addition, levels were in the rescued in the Utrn-/- × mdx mice, levels to levels in × Utrn-/- × mdx and × Utrn-/- × mdx were and expression the hDMD transgene not reverse the of dystrophic gene expression in dystrophic RT-PCR and expression profiling experiments. In a identified a of for which the expression with the of muscular R. Sterrenburg E. van de Meijer E.J. de S. Campbell K.P. S. van Ommen G.J. den Dunnen J.T. Hoen't P.A. J. 2006; PubMed Scopus Google Scholar). the expression of dystrophy in extracellular matrix and not between × Utrn-/- × × Utrn-/- × mdx and mice We used to muscular expression of mice the hDMD gene on an mdx or Utrn-/- × mdx In the expression of a of that are in dystrophic mdx mice and Utrn-/- × mdx mice. of these in as cardiac and have been to be in expression profiling studies in mdx mice R. Sterrenburg E. de Meijer E.J. van Ommen G.J. den Dunnen J.T. Hoen't P.A. BMC Genomics. 2005; 6: 98Crossref PubMed Scopus (83) Google Scholar, A.P. S. Hum. Mol. Genet. 2003; 12: PubMed Scopus Google and DMD M. Chen J. Hoffman E.P. 12: Full Text Full Text PDF PubMed Scopus Google Scholar, R. Hoffman E.P. J. Biol. PubMed Scopus Google Scholar, D. S.A. Kunkel L.M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). it is that expression levels for the in dystrophic from mdx and mdx × Utrn-/- mice to levels in for dystrophin the human dystrophin gene. Expression levels of in dystrophic in are by expression of the hDMD a of 1 × were found expressed between mice and the rescued mdx and mdx × Utrn-/- mice of these has been with muscular dystrophy and were not located in the on chromosome the hDMD gene is from expression profiling in found for in gene expression levels of in chromosomal these that expression of the hDMD gene the expression of and In the show that of yeast spheroplasts with embryonic stem cells is a for the of functional human DNA to the mice with the full-length hDMD transgene a much and functional of human DNA integrated into a mouse chromosome than thus A mouse with an stretch of DNA of the human chromosome has been A. S. C. S. A. S. L. D. J.M. S. D.J. D. Fisher Science. 2005; PubMed Scopus Google Scholar). This of DNA as a which has a of and In hDMD gene stable The expression of full-length hDMD transcripts and the 427-kDa human dystrophin protein were confirmed by expression histological and Western blotting. an proof for its the expression of the hDMD at levels to the levels of the endogenous mouse dystrophin in mice, rescued the dystrophic phenotype of mdx and mdx × Utrn-/- mice. This was not at the from and profiling of the expression of a highly muscular gene R. Sterrenburg E. van de Meijer E.J. de S. Campbell K.P. S. van Ommen G.J. den Dunnen J.T. Hoen't P.A. J. 2006; PubMed Scopus Google Scholar). with the of the mdx phenotype by of and containing hDMD C. J.M. D. G. C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, R. J. S. Tinsley J.M. A. Davies K.E. G. Hum. Gene Ther. PubMed Scopus Google Scholar, A. H. K.E. Dickson G. D.J. Gene Ther. 2004; PubMed Scopus Google Scholar, S. S. Chen Campbell K.P. Gene Ther. Google Scholar). the human dystrophin which has 91% amino acid identity with mouse is to its counterpart. This a of the protein and In the hDMD for the that for splicing and of the dystrophin are conserved between and mice. The tissue-specific of the expression of dystrophin transcripts to be for the hDMD transgene in the We differences between the expression from the transgene and the endogenous Dmd gene. The human Dp427p and Dp427m promoters to be more than their endogenous the reverse is for Dp427c and This be to the human which are not the transcription it be to the different genomic and in which the and the human DMD are The hDMD mouse model in the the and of the different promoters in the DMD gene. In × mdx × Utrn-/- mice. In is an to a of mdx × Utrn-/- mice, an model for DMD and The hDMD mouse is of for the and of human sequence-specific therapeutics for exon with the of reading frame is a highly to the DMD phenotype into the milder Becker most in with AONs were in the mdx exon 23 was Q.L. Mann C.J. Lou F. Bou-Gharios G. Morris G.E. Xue S.A. Fletcher S. Partridge T.A. Wilton S.D. Nat. Med. 2003; 9: 1009-1014Crossref PubMed Scopus (327) Google Scholar, 19Lu Q.L. Rabinowitz A. Chen Y.C. Yokota T. Yin H. Alter J. Jadoon A. Bou-Gharios G. Partridge T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 198-203Crossref PubMed Scopus (343) Google Scholar, 20Mann C.J. Honeyman K. Cheng A.J. Ly T. Lloyd F. Fletcher S. Morgan J.E. Partridge T.A. Wilton S.D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 42-47Crossref PubMed Google Scholar, 21Alter J. Lou F. Rabinowitz A. Yin H. Rosenfeld J. Wilton S.D. Partridge T.A. Lu Q.L. Nat. Med. 2006; 12: 175-177Crossref PubMed Scopus (429) Google Scholar, 22Goyenvalle A. Vulin A. Fougerousse F. Leturcq F. Kaplan J.C. Garcia L. Danos O. Science. 2004; 306: 1796-1799Crossref PubMed Scopus (400) Google Scholar). In the hDMD it is to the exon of human AONs with the as be therapeutic for DMD M. A. de Meijer E.J. Kaman W.E. Janson A.A. R.H. van Ommen G.J. den Dunnen J.T. van Deutekom J.C. Mol. Ther. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). for DMD that at gene correction C. T.A. Hum. Gene Ther. 13: PubMed Scopus Google in testing of with the that be therapeutic for the in DMD be to in exon therapy in the of the DMD of Becker the deletion have been described, their clinical and these life are Becker have been In these it be to study in in for dystrophin are not available. These be by of the exon in the hDMD × mdx the be by the of the full-length hDMD A be to or deletions in the or in the cells containing the hDMD gene. the it be to transgenic mutation in the DMD gene. can be for their on an mdx or mdx × Utrn-/- In addition, deletions to found in DMD can be Crossing of mice with these with mdx or mdx × Utrn-/- mice study of the therapeutic of human AONs in the most The transgenic hDMD mouse model can be used for more The of deletion in the hDMD gene is (1den Dunnen J.T. Bakker E. van Ommen G.J. Pearson P.L. Br. Med. Bull. 1989; 45: 644-658Crossref PubMed Scopus (13) Google Scholar, D.R. A. Roche Davies K.E. Genomics. 1991; PubMed Scopus Google Scholar). The for is in the chromosomal context more or and recombination Since not to M. U. R. R. A. G. N. Hum. Genet. 2003; PubMed Scopus Google it is that as influence the of genomic in the different of the dystrophin gene. The hDMD mouse full-length dystrophin in different genomic be to the of in cells is by the chromosomal This of study be in of and cells from hDMD mice and be by the of in the different mutation In the hDMD mouse is a model to the of DNA in the mouse genome and its in and gene therapeutic studies for We highly by van Kaman Leiden and We the Leiden for and van Leiden are acknowledged for on the We are to Leiden and C. of of mouse embryonic stem with