Optimisation of growth hormone production by muscle cells using plasmid DNA

Abstract

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.

Modifying muscle mass – the endocrine perspective

“…MacColl et al (2000) successfully employed this approach using GH as the target. The C2C12 model of murine muscle cells was used and GH secretion was measurable under several transfection conditions, including various stages of cellular differentiation into syncitial myotubes.…”

IGF1 gene transfer into skeletal muscle using recombinant adeno-associated virus in a rat model of liver cirrhosis

“…rAA V production and characterisation> The IG F1 expression cassette includes rat IGFI cDNA (kind gift from P. Rotwein) driven by the CMV promoter, the bovine growth hormone polyadenylation site from pCDNA3 (Invitrogen) and the myosin light chain enhancer 1/3 (12,17). Plasmid pAAVIGF1 contains the IGF1 expression cassette flanked by AA V ITRs, and was created by ligation of the expression cassette into Sal I-digested pAAV.LucA (kind gift from I. Maxwell).…”

Therapeutic Angiogenesis: Foundations and Practical Application

“…The most widely spread vectors include adeno- [38], adeno-associated [39] and retroviruses, yet in therapeutic angiogenesis, the later have limited application due to high risk of insertional mutagenesis [40]. Recent progress of molecular engineering allowed development of optimized viral systems exploiting their advantages as well as novel more efective pDNA systems [41,42].…”