Characterization and genetic manipulation of porcine muscle cells for ex vivo cell-mediated gene transfer

John Russell Blanton, Purdue University

Abstract

Successful myoblast-mediated gene transfer requires the development of methods for incorporating engineered myoblasts into muscle. The objective of these studies were (1) develop techniques for the rapid transformation of porcine muscle cells with marker genes; (2) develop techniques for isolation of myoblasts from mixed cell preparations and (3) investigate the effects of notexin administration and immunosuppression on myoblast-mediated gene transfer. Porcine muscle cells were clonally isolated from male pigs. Myogenic lineage was determined by muscle-specific antibodies, myotube fusion assays and reverse transcriptase-PCR for muscle-specific mRNA. Cationic liposomes and electroporation were tested for their ability to transfect muscle cells. LipofectAMINE resulted in the greatest (P < .01) transient luciferase expression when compared to the other methods. Stable electroporations, and lipofectAMINE transfections using green fluorescence protein (GFP) were conducted. LipofectAMINE efficiencies were 1:16,000 for myoblasts and 1:33,000 for fibroblasts. Optimal stable electroporation parameters resulted in efficiencies of 1 × 10 −5 cells for myoblasts and fibroblasts. Transduction of muscle cells with the vesicular stomatitis virus glycoprotein-G pseudotyped retrovirus resulted in efficiencies of 1:1.2 for myoblasts and 1:1.1 for fibroblasts. In order to rapidly isolate myoblasts, cell were characterized by flow cytometry using forward angle light scatter (FALS), ninety-degree light scatter (90°LS) and a fluorescein-labeled myoblast-specific antibody (5.1H11). No differences in FALS and 90°LS were detected between clonal myoblasts and fibroblasts, however primary muscle cell preparations revealed two cell populations based on FALS. Small cell populations were found to be >90% myoblasts, whereas the larger cell population was 64% myoblasts. Population size differences were lost following second passage. When myoblasts were sorted using 5.1H11, >90% were desmin positive. Transduced myoblasts and fibroblasts were separated and implanted. On day 4, implanted muscles were removed and GFP protein and retroviral sequences were detected in fibroblast injection sites. Immune suppression did not effect implantation success, however, GFP was only detected in sited that received notexin. In conclusion, cationic liposome are superior to electroporation for transient transfection, but retroviral transduction is most efficient at producing stable expression. Porcine myoblasts can be isolated from mixed primary preparations using 5.1H11 or FALS. Myoblast implantation resulted in GFP expression in one animal, whereas, implanted fibroblasts were detected in 2 animals. These results indicate that successful myoblast-mediated gene transfer may be used in the pig to investigate muscle growth and development.

Degree

Ph.D.

Advisors

Bidwell, Purdue University.

Subject Area

Livestock|Molecular biology|Veterinary services

COinS