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 a pproach for studying roles of locally produced ICF-I on IGF-binding protein (IGFBP) production. To determine parameters for maximal reporter gene expr ession, and to investigate the effects of dose, time and weaning on exogeno us DNA expression, plasmid DNA encoding firefly luciferase under control of a constitutive promoter and enhancer was injected ill skeletal muscle of p igs, Results indicate that injected DNA does not migrate beyond 9 mm from i njection sites and that 100 mu g DNA injections resulted in optimal lucifer ase activity. Maximum amounts of recombinant protein were observed 3 days a fter injection, and were reduced by weaning. Using these data, a second DNA injection study was performed using plasmid DNA containing a cDNA insert f or epitope-tagged insulin-like growth factor-I (TIGF-I). Significant quanti ties of TIGF-I were detected by ELISA and confirmed by western blotting. Bo th 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 ap proach of gene transfer results in biologically active recombinant protein production in pot-cine skeletal muscle, and provides an excellent in vivo m odel for studying the autocrine and (or) paracrine effects of locally produ ced growth factors in skeletal muscle.