Full length article Electrotransfer of the epinecidin-1 gene into skeletal muscle enhances the antibacterial and immunomodulatory functions of a marine fish, grouper (Epinephelus coioides) Lin-Han Lee a , Cho-Fat Hui b , Chi-Mu Chuang c, d , Jyh-Yih Chen a, * a Marine Research Station, Institute of Cellular and Organismic Biology, Academia Sinica, 23-10 Dahuen Road, Jiaushi, Ilan 262, Taiwan b Institute of Cellular and Organismic Biology, Academia Sinica,128 Academia Road, Section 2, Nankang, Taipei 115, Taiwan c Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Taipei Veterans General Hospital, Taipei, Taiwan d Faculty of Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan article info Article history: Received 14 June 2013 Received in revised form 30 July 2013 Accepted 30 July 2013 Available online 23 August 2013 Keywords: Epinecidin-1 Electroporation Epinephelus coioides Vibrio vulnificus abstract Electrotransfer of plasmid DNA into skeletal muscle is a common non-viral delivery system for the study of gene function and for gene therapy. However, the effects of epinecidin-1 (epi) on bacterial growth and immune system modulation following its electrotransfer into the muscle of grouper (Epinephelus coioides), a marine fish species, have not been addressed. In this study, pCMV-gfp-epi plasmid was electroporated into grouper muscle, and its effect on subsequent infection with Vibrio vulnificus was examined. Over-expression of epi efficiently reduced bacterial numbers at 24 and 48 h after infection, and augmented the expression of immune-related genes in muscle and liver, inducing a moderate innate immune response associated with pro-inflammatory infiltration. Furthermore, electroporation of pCMV- gfp-epi plasmid without V. vulnificus infection induced moderate expression of certain immune-related genes, particularly innate immune genes. These data suggest that electroporation-mediated gene transfer of epi into the muscle of grouper may hold potential as an antimicrobial therapy for pathogen infection in marine fish. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction The delivery of plasmid DNA encoding functional protein via injection into skeletal muscle has the potential to treat an extensive range of diseases [1e3]. The factors affecting gene transfer effi- ciency and recombinant protein expression following gene delivery include: (i) the system of delivery, (ii) the immune response to the plasmid or transgenic protein, and (iii) non-specific expression of the transgene in non-muscle cells. The electroporation method of delivering plasmid DNA is based on the application of an electric field, to both enhance cell permeability and provide an electro- phoretic force to introduce the plasmid DNA [4]. Electroporation enhances uptake of plasmid DNA by skeletal muscle by more than 2000-fold, as compared to injection of plasmid DNA alone; furthermore, expression of exogenous DNA can persist for up to one year [5]. Enormous improvements in the technology and trans- duction efficiency in mammalian systems have been made over the past decade, and it has now been successfully used in pre-clinical and clinical trials for the transfer of therapeutic plasmids and DNA vaccines [6e8]. Intra-muscular injection of plasmid DNA has been studied in several fish species, including carp, tilapia, goldfish, zebrafish, and flounder [9]. However, to date there are few reports on the elec- troporation of fish muscle [10e13]. Intra-muscular injection com- bined with electrotransfer of plasmid DNA has only thus far been reported for zebrafish and European seabass (Dicentrarchus labrax) [10,11]. The efficiency of gene transfer following intra-muscular injection of plasmid DNA is relatively poor in marine as compared to freshwater fish, on account of differences in muscle plasma osmolality and ion concentrations. It has been shown that transfection efficiency in a mammalian system can be enhanced by using a high voltage electric pulse [14], but differences in plasma osmolality may make this unsuitable for marine fish. Antimicrobial peptides (AMPs) are key components of innate immunity in all multicellular organisms, and their bactericidal properties have been extensively studied [15,16]. Their mechanism of action involves permeation of bacterial membranes, which is presumed to reduce the possibility of the emergence of AMP * Corresponding author. Tel.: þ886 920802111; fax: þ886 39871035. E-mail address: zoocjy@gate.sinica.edu.tw (J.-Y. Chen). Contents lists available at ScienceDirect Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi 1050-4648/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fsi.2013.07.050 Fish & Shellfish Immunology 35 (2013) 1359e1368