The effectiveness of adenoviral vectors to deliver and express genes in rainbow trout, Oncorhynchus mykiss (Walbaum) K Overturf 1 , S LaPatra 2 and P N Reynolds 3 1 USDA/ARS, Hagerman Fish Culture Experiment Station, Hagerman, ID, USA 2 Clear Springs Foods, Inc., Research Division, Buhl, ID, USA 3 Division of Human Gene Therapy, Departments of Medicine, Surgery and Pathology and Gene Therapy Center, University of Alabama at Birmingham, AL, USA Abstract The efficacy of adenoviral vectors for gene delivery into fish cells, both in vitro and in vivo, was eval- uated. Vectors utilized were of human adenovirus serotype 5 (Ad), which are commonly used in human clinical trials, but have not been assessed for gene delivery to fish. Because nothing is known about Ad receptors in fish, both an Ad (Ad5Luc1) with natural tropism for the coxsackie and adeno- virus receptor (CAR), as well as an infectivity enhanced Ad (Ad5LucRGD) were included within this study. Gene expression was detected in cell lines using either vector. The levels seen with Ad5Luc- RGD were much higher than for Ad5Luc1 in most lines except CHSE-214. Transduction of CHSE- 214 cells with Ad5Luc1 could be blocked with an excess of a competitive inhibitor, suggesting that these cells possess a CAR homologue that mediates attachment of Ad, similar to that seen in mamma- lian cells. In vivo gene delivery was attempted by several methods, with significant expression seen only via intramuscular injection, although infection efficiency was low. Thus it was observed that several teleost cell lines are capable of being infected and one cell line expressed a human serotype adenoviral receptor homologue that aids in Ad infection. Additionally, in vivo studies indicated that muscle tissue of rainbow trout could be infected with Ad vectors, suggesting an alternative gene delivery strategy for this animal. Keywords: adenoviral vectors, gene delivery, Onc- orhynchus mykiss, rainbow trout. Introduction Currently, transfer of genetic material, whether for targeted gene expression or for DNA vaccines, is limited in fish. Strategies assessed to date include the use of various physical methods, including injection (Lorenzen, Lorenzen, Einer-Jensen & LaPatra 2002), particle bombardment (Zelenin, Alimov, Barmintzev, Beniumov, Zelenina, Krasnov & Kolesnikov 1991), chemical transfection reagents (Sussman 2001) and electroporation (Muller, Lele, Varadi, Menczel & Orban 1993) into fish eggs. Transduction into fish muscle tissue has also been achieved using particle bombardment with a gene gun (Lee, Hirono & Aoki 2000). Limited studies of in vitro gene delivery to fish cells in culture have been reported using vesicular stomatitis glycopro- tein (VSVG)-pseudotyped retrovirus (Burns, Fried- mann, Driever, Burrascono & Yee 1993) and Semliki forest virus (Phenix, McKenna, Fitzpatrick, Vaughan, Atkins, Liljestrom & Todd 2000). Nev- ertheless, a system of broad utility for the delivery of genes into juvenile or adult fish in vivo has not been established. Research into the development of gene therapy approaches for human disease has been associated with a dramatic increase in the range of gene delivery strategies available for achieving in vivo Journal of Fish Diseases 2003, 26, 91–101 Correspondence Dr K Overturf, USDA/ARS, Hagerman Fish Culture Experiment Station, 3059-F National Fish Hatchery Road, Hagerman, ID 83332, USA (e-mail: kennetho@uidaho.edu) 91 Ó 2003 Blackwell Publishing Ltd