Optimisation of growth hormone production by muscle cells using plasmid DNA.
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.
MacColl
G S
GS
Department of Medicine, Royal Free and University College Medical School, London, UK. gmaccoll@rfc.ucl.ac.uk
Novo
F J
FJ
Marshall
N J
NJ
Waters
M
M
Goldspink
G
G
Bouloux
P M
PM
D000818:Animals; D001681:Biological Assay / Q000379:methods; D015153:Blotting, Western; D002478:Cells, Cultured; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage*; D013006:Growth Hormone / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism*; D010957:Plasmids; D011817:Rabbits; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014162:Transfection / Q000379:methods*
D013006:Growth Hormone