HORIZONTAL GENE TRANSFER IN PIG SKELETAL MUSCLE

¹ Department of Animal Sciences, Purdue University, West Lafayette, IN 47907-1026 USA

² Department of Biological Sciences, Purdue University, West Lafayette, IN 47907 USA

³ Basic Medical Sciences, Purdue University, West Lafayette, IN 47907 USA

⁴ Department of Animal and Range Science, South Dakota State University, Brookings, SD 57007 USA

DNA injection and cell-mediated gene transfer methods are being developed in the pig to enable gene regulation studies in skeletal muscle fibers. In vivo models are necessary because development cannot proceed beyond the myotube stage in cell culture. DNA injection studies were conducted to determine the effect of DNA dose, time and weaning stress on luciferase reporter gene expression. Luciferase activity could be detected for at least 21 days post-injection and was sample site dependent (P<.0001). Beyond 9 mm from injection sites, the amount of luciferase was less than 1% of that found at injection sites. Weaning had a negative effect on exogenous gene expression as observed by 3-fold lower (P<.0001) amounts of luciferase activity in weaned pigs relative to non-weaned pigs. Cell-mediated gene transfer requires methods to isolate myoblasts that retain their proliferative capacity. A human monoclonal antibody was identified for separation of myoblasts from non-myoblasts by fluorescence-activated cell sorting (FACS) of primary muscle cell cultures. Porcine muscle cells were transduced with a green fluorescence protein (GFP) reporter using stomatitis virus glycoprotein G pseudotyped retrovirus. Transduced GFP-positive cells were separated from GFP-negative cells by FACS. Expression of GFP was stable for six doublings as cells were proliferated for re-implantation. Treatments for immune suppression (FK506) and induced muscle repair (Notexin) were tested in conjuction with cell-mediated gene transfer. Fibroblasts expressing GFP could be readily detected at injection sites and fusion of myoblasts to muscle fibers at a single injection site was confirmed by detection of GFP within the fibers.