Factors affecting production of luciferase and epitope-tagged IGF-I in porcine muscle after DNA injection.
Direct DNA injection into porcine skeletal muscle was investigated as an approach for studying roles of locally produced IGF-I on IGF-binding protein (IGFBP) production. To determine parameters for maximal reporter gene expression, and to investigate the effects of dose, time and weaning on exogenous DNA expression, plasmid DNA encoding firefly luciferase under control of a constitutive promoter and enhancer was injected in skeletal muscle of pigs. Results indicate that injected DNA does not migrate beyond 9 mm from injection sites and that 100 microg DNA injections resulted in optimal luciferase activity. Maximum amounts of recombinant protein were observed 3 days after injection, and were reduced by weaning. Using these data, a second DNA injection study was performed using plasmid DNA containing a cDNA insert for epitope-tagged insulin-like growth factor-I (TIGF-I). Significant quantities of TIGF-I were detected by ELISA and confirmed by western blotting. Both IGFBP-2 and IGFBP-2 mRNA were increased in treated muscle compared with controls. We conclude that increased expression of IGF-I in muscle results in increased IGFBP-2. Furthermore, these data indicate that this in vivo approach of gene transfer results in biologically active recombinant protein production in porcine skeletal muscle, and provides an excellent in vivo model for studying the autocrine and (or) paracrine effects of locally produced growth factors in skeletal muscle.
Everett
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RS
Department of Animal Sciences, Purdue University, West Lafayette, Indiana 47907-1151, USA.
Gerrard
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DE
Grant
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D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western / Q000379:methods; D004247:DNA / Q000008:administration & dosage*; D003720:Densitometry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D000939:Epitopes; D015870:Gene Expression; D018014:Gene Transfer Techniques; D007091:Image Processing, Computer-Assisted; D007273:Injections, Intramuscular; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000096:biosynthesis / Q000235:genetics*; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics*; D008954:Models, Biological; D018482:Muscle, Skeletal / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine; D013997:Time Factors; D014886:Weaning
D000939:Epitopes; D018971:Insulin-Like Growth Factor Binding Protein 2; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D004247:DNA; D008156:Luciferases