Direct DNA injections for the production of recombinant proteins in porcine skeletal muscle
Abstract
In vivo transfection of porcine skeletal muscle by intramuscular injections of purified plasmid DNA containing a reporter gene is a simple and efficient alternative to the ex vivo method of myoblast-mediated gene transfer. These studies were performed to develop an in vivo muscle injection assay for quantifying reporter gene expression levels, analyzing exogenous DNA migration and the effects of DNA dose, time and weaning on recombinant protein production. Experiments were also conducted to determine the biological activity of the recombinant protein. To determine parameters for maximal expression of a reporter gene, and to investigate the effects of dose, time and weaning on exogenous DNA expression in porcine skeletal muscle, plasmid pGL3C DNA containing firefly luciferase cDNA under control of an eukaryotic SV40 promoter and enhancer was injected into the longissimus muscle of pigs. Luciferase activity was assayed in muscle samples using a luminometer. Results indicate that injected DNA does not migrate beyond 9mm on either side of injection site and 100 μg DNA injections resulted in highest luciferase activity (P < .0001). Maximum levels of recombinant protein were observed at 3d post-injection (P < .01) and weaning significantly reduced (P < .0001) exogenous DNA expression. A second study using plasmid pcDNA3.1(+) with a cDNA insert for tagged insulin-like growth factor-I (TIGF-I) was conducted using porcine skeletal muscle. Recombinant IGF-I was quantified by ELISA, and localized by immunohistochemistry. Its immunoreactivity with both the T7 and IGF-I antibodies was confirmed by western blotting and immunohistochemistry. Its biological activity was determined by its ability to increase levels of insulin-like growth factor binding protein-2 (IGFBP-2) mRNA in injected muscle. A significant increase (P < .0001) was observed in IGFBP-2 mRNA in treated muscle as compared with controls. Increases in IGFBP-2 protein in treated muscles were detected by ligand blotting. Persistence of injected plasmid was detected by Southern blotting. These data indicate that this in vivo approach of direct DNA injections can result in biologically active recombinant protein production in porcine skeletal muscle, and provides an excellent model for studying the autocrine, and/or paracrine, effects of locally produced IGF-I and other growth factors in skeletal muscle.
Degree
Ph.D.
Advisors
Grant, Purdue University.
Subject Area
Livestock|Anatomy & physiology|Animals|Molecular biology
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