title: Epitope-tagged insulin-like growth factor-I expression in muscle.
abstract: Development of a recombinant insulin like growth factor I (IGF-I) that is distinguishable from its endogenous counterpart would provide a powerful tool for delineating the role of IGF in myogenesis. Therefore, the objective of this study was to create an epitope-tagged IGF-I that retains biological activity and determine whether expression of this construct is possible in muscle tissue following direct DNA injection. Expression vectors were created that encoded porcine IGF-I containing a T7 (11-amino acid) epitope-tag (TIGF). Immunoreactivity of the purified recombinant TIGF was confirmed using monoclonal antibodies. Biological activity was evaluated by examining differentiation of myoblasts cultured with TIGF or transfected with TIGF plasmid DNA. Addition of purified TIGF to myoblast cultures stimulated (P < 0.05) muscle creatine kinase levels similar to insulin (10(-5) M). Likewise, transfection of L6A1 with TIGF DNA hastened (P < 0.01) differentiation compared to control pcDNA-transfected myoblasts. The integrity of the recombinant protein was confirmed using a sandwich-configured enzyme linked immunosorbent assay. Finally, recombinant TIGF DNA was injected in porcine muscle and the ability to detect TIGF protein was evaluated. TIGF expression was detected in muscle fibers of injected porcine muscle. These data show that a T7 amino acid tag placed on the amino terminus of the IGF-I protein remains intact during processing and does not interfere with the biological activity of the molecule. Use of this DNA construct is an excellent tool for investigating the role of IGFs in control muscle development and provides a model to investigate other regulators of animal growth.
authors.affiliation: Department of Animal Sciences, 1151 Smith Hall, Rm. 202A, Purdue University, West Lafayette, IN 47907-1151, USA.
authors.forename: C L
authors.initials: CL
authors.lastname: Reichel
authors.forename: A L
authors.initials: AL
authors.lastname: Grant
authors.forename: R S
authors.initials: RS
authors.lastname: Everett
authors.forename: C A
authors.initials: CA
authors.lastname: Bidwell
authors.forename: D E
authors.initials: DE
authors.lastname: Gerrard
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western / Q000662:veterinary; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000662:veterinary; D003402:Creatine Kinase / Q000032:analysis; D017931:DNA Primers / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D000939:Epitopes; D005786:Gene Expression Regulation; D007273:Injections, Intramuscular / Q000662:veterinary; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D008856:Microscopy, Fluorescence / Q000662:veterinary; D018482:Muscle, Skeletal / Q000276:immunology / Q000502:physiology*; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000662:veterinary; D013552:Swine / Q000276:immunology / Q000502:physiology*; D014162:Transfection / Q000235:genetics
chemical_list: D000911:Antibodies, Monoclonal; D017931:DNA Primers; D000939:Epitopes; D011994:Recombinant Proteins; D007334:Insulin-Like Growth Factor I; D003402:Creatine Kinase