Myosin regulatory elements as vectors for gene transfer by intramuscular injection

Skarli, M.; Kiri, Arpna; Vrbová, Gerta; Lee, Ca; Goldspink, Geoffrey

doi:10.1038/sj.gt.3300618

Cited by 23 publications

(12 citation statements)

References 16 publications

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“…This vector used a truncated myosin heavy chain promoter in combination with the myosin light chain enhancer element, and the results were consistent with previous observations where these vectors were used for the expression of human alpha-galactosidase (Novo et al 1997). However, we have also shown that the promoter elements used in pMHLC-GH generate tissue-specific gene expression in muscle fibres (Skarli et al 1998) and as other studies show that specific types of muscle promoters are effective at producing hormones in muscle cells (Barton-Davis et al 1998, Li et al 1999, this vector is being studied further.…”

Section: Discussionsupporting

confidence: 79%

Optimisation of growth hormone production by muscle cells using plasmid DNA

MacColl¹,

Novo²,

Marshall³

et al. 2000

Journal of Endocrinology

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The production of peptide hormones by skeletal muscle tissue is a promising area of gene therapy. Skeletal muscle myogenesis can be induced in vitro, resulting in the fusion of mononucleate myoblasts to form multinucleate myotubes, and delivery vectors are first tested in vitro. C2C12 myoblasts transfected with pcDNA3-GH, which used the human cytomegalovirus (CMV) promoter, secreted immunoreactive GH with comparable biological activity to pituitary GH. Mouse myeloid leukaemia cells, which express the mouse GH receptor were used for the bioassay, and activation of these cells by GH was measured by a colorimetric microculture tetrazolium assay. Cells were incubated with a tetrazolium salt (MTS) and an intermediate electron acceptor (phenazine methosulphate, PMS), and formazan production was measured as optical density (O.D.) at 490 nm.The efficiencies of several plasmid expression vectors were compared in differentiated and non-differentiated muscle cells, as a function of bioactive GH secreted by the transfected cells. Ten-day differentiated C2C12 myotubes transfected with pcDNA3E-GH, which used the CMV promoter and a rat myosin light chain enhancer element, secreted significantly more biologically active GH than myotubes transfected with pcDNA3-GH (0·82 O.D. units 0·06 vs 0·57 0·05 respectively, P<0·001). This was consistent with reduced CMV promoter activity in myotubes. Myoblasts transfected with pcDNA3-GH secreted more bioactive GH than 10-day transfected myotubes (1·1 0·1 vs 0·77 0·07 respectively). However, the responses were indistinguishable (both 1·0 0·09) if both the myotubes and myoblasts had been transfected with pcDNA3E-GH. Substitution of the vector pMHLC-GH, which used a muscle-specific truncated rabbit myosin heavy chain promoter, and the myosin enhancer resulted in a marked decrease in the responses to the conditioned medium from fused myotubes compared with the vectors pcDNA3-GH and pcDNA3E-GH (0·24 0·02 vs 0·57 0·05 vs 0·82 0·06 respectively). We concluded that the combination of CMV promoter and myosin light chain enhancer in pcDNA3E-GH had the greatest expression efficiency of the several plasmid vectors which we investigated.

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Section: Discussionsupporting

confidence: 79%

Optimisation of growth hormone production by muscle cells using plasmid DNA

MacColl¹,

Novo²,

Marshall³

et al. 2000

Journal of Endocrinology

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“…The latter type of DPM is sometimes interpreted as myofibers with overproduction of dystrophin. 17,18 Meanwhile histopathological changes in csDPM (mononuclear infiltration, great variations in crosssectional area with unusually small cross-sections of some fibers, fragmentation of some csDPM, and the absence of contact with neighboring fibers) observed after ballistic transfection, SOC vector delivery and after maximal dosages of MF-2 constructs tested in this study, provide evidence for their interpretation as degenerative myofibers. 10,11 It has been postulated that overexpression of transgenic dystrophin corrects dystrophic symptoms in mdx mice without causing deleterious side-effects.…”

Section: Discussionsupporting

confidence: 58%

“…18 A significant increase of DPM count in the mdx muscles not subjected to gene transfection was first reported in our studies with ballistic transfection and synthetic oligopeptide complexes. 10,11 Full-length dystrophin cDNA constructs (pHSADy) delivered by these vectors to the right quadriceps femoris muscle resulted in 10-to 20-fold elevation of DPM score in the muscles of the opposite limb.…”

Section: Figure 6 Relative Numbers Of MDX Myofiber Nuclei Containing supporting

confidence: 62%

Local and distant transfection of mdx muscle fibers with dystrophin and LacZ genes delivered in vivo by synthetic microspheres

et al. 1999

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Patterns of dystrophin and ␤-galactosidase expression ticles were detected by FISH analysis in about 60-70% of were examined in mdx mice after i.m. injections of synmyofiber nuclei in muscles of injected and contralateral thetic microspheres (MF-2) loaded with full-length limbs 7 days after application. The presence of human dys-(pHSADy) or mini-dystrophin gene (pSG5dys) cDNA plastrophin cDNA and its products in all skeletal muscles and mid constructs or with LacZ marker gene (pCMV-LacZ). A in different internal organs was proven by PCR and RTsingle injection of 25 g pHSADy into quadriceps femoris PCR analysis. Patches of ␤-galactosidase expression were muscle resulted in 6.8% of dystrophin positive myofibers abundant in injected muscle, and frequent in the contralat-(DPM) in a given muscle; 8.4% of DPM in glutaeus muscle eral and other skeletal muscles as well as in diaphragm, and 4.3% of DPM in quadriceps femoris muscle of contraheart and lungs. High levels of dystrophin cDNA lateral limb on day 21 after exposure compared with only expression, and an efficient distant transfection effect with 0.6% DPM in intact (non-injected) mdx mice. A high propreferential intranuclei inclusion of MF-2 vehicle, are very portion of DPM (17.6% and 10.8%, respectively) was regisencouraging for the development of a new constructive tered in both injected and contralateral muscles after ministrategy in gene therapy trials of DMD. gene cDNA administration. MF-2/dystrophin cDNA par-

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“…Some studies have investigated the number of muscle-specific promoters, such as skeletal muscle α-action, muscle creatine kinase (MCK), myosin heavy chain, and so on. The relationship between the promoters and the muscle proliferation and transcriptional activity of these promoters has also been investigated (Skarli et al, 1998;Hagstrom et al, 2000;Zhu et al, 2005). In this study, we showed that the promoter and core promoter of CMYA1 are muscle-specific promoters.…”

Section: Introductionmentioning

confidence: 50%

Promoter analysis of bovine cardiomyopathy-associated protein 1 gene

et al. 2016

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ABSTRACT. The CMYA1 (cardiomyopathy-associated protein 1) is an actin-binding protein that plays a vital role in cardiac morphogenesis. CMYA1 is expressed specifically in the myocardial and skeletal muscle and is up-regulated in injured muscle. We therefore speculated that the bovine CMYA1 promoter might be muscle-specific. In this study, the promoter (+20/-1135) region of the bovine CMYA1 gene was cloned into a pEGFP-1 vector, and we found that the EGFP was observed only in C2C12 and myoblast cells. Thus, the CMYA1 promoter is musclespecific. Thereafter, eight pGL3-basic vectors with various truncated CMYA1 promoter fragments were transfected into C2C12 cells, to identify the core promoter region using a Dual-Luciferase Reporter Assay System. The results showed that the promoter region from -457 to +20 bp was essential for CMYA1 to maintain the promoter activity, implying that this region may be the CMYA1 core promoter. We thus illustrate that the core promoter is muscle-specific. To evaluate the activity of the CMYA1 core promoter, the CMYA1 core and muscle creatine kinase (MCK) promoters were cloned into a pcDNA3.1 vector. The expression levels of their target genes were measured in C2C12 cells using real-time polymerase chain reaction. The CMYA1 promoter drove the expression of the target gene six times higher than did the MCK promoter. The results thus suggest that the CMYA1 promoter could be an effective muscle-specific promoter, which may be useful in further studies of cardiomyopathy treatment and transgenic animal research.

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Myosin regulatory elements as vectors for gene transfer by intramuscular injection

Cited by 23 publications

References 16 publications

Optimisation of growth hormone production by muscle cells using plasmid DNA

Optimisation of growth hormone production by muscle cells using plasmid DNA

Local and distant transfection of mdx muscle fibers with dystrophin and LacZ genes delivered in vivo by synthetic microspheres

Promoter analysis of bovine cardiomyopathy-associated protein 1 gene

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