DNA Transfer Into Fish Bone Cells 51 MOLECULAR BIOTECHNOLOGY Volume 34, 2006 RESEARCH PROTOCOLS 51 Molecular Biotechnology © 2006 Humana Press Inc. All rights of any nature whatsoever reserved. ISSN: 1073–6085/Online ISSN: 1559–0305/2006/34:X/000–000/$30.00 *Author to whom all correspondence and reprint requests should be addressed. *Author to whom all correspondence and reprint requests should be addressed. Centre of Marine Sciences (CCMAR), University of Algarve, Campus de Gambelas, 8005-139 Faro, Portugal. E-mail: vlaize@ualg.pt. Abstract Enhanced DNA Transfer Into Fish Bone Cells Using Polyethylenimine Daniel Braga, Vincent Laizé,* Daniel M. Tiago, and M. Leonor Cancela The use of in vitro cell culture systems to assess gene function largely depends on the successful transfer of DNA into target cells. Well developed in mammals, transfection methods are still to be optimized for non-mammalian cell culture systems, like fish. Here we describe a rapid, cost-efficient, and successful method to transfer DNA into a fish bone-derived cell line using polyethylenimine (PEI) as the DNA carrier. Using this method, DNA transfer was remarkably enhanced in comparison with commercially available reagents, as demonstrated by the increased activity of both luciferase and green fluorescent protein observed in the transfected cells. Its efficiency in transferring DNA into a wide range of cell types, including non-mamma- lian and hard-to-transfect cells, in addition to a low cost, show that PEI is a reagent of choice for nonviral vector transfection. Index entries: DNA transfer; cell transfection; polyethylenimine (PEI); non-mammalian cells. 1. Introduction In the last few years, there has been a huge increase in available genomic information result- ing from whole-genome sequence analysis of many different organisms. Bioinformatics and in silico comparative genomic analysis have iden- tified putative functions for many previously unknown genes. However, this must be comple- mented by functional analysis, an approach that often requires the use of in vitro cell culture sys- tems. Because it offers technical advantages over other vertebrates, fish have recently become a model animal for studying skeletal development (1,2). Fish bone-derived cells have been recently developed and characterized (3) but the produc- tion of new cell clones derived from these cell lines depends on the successful incorporation of DNA into target cells and is therefore limited by the development/optimization of DNA carriers able to promote this delivery. This is particularly important for bone-derived cells that are notori- ously difficult to transfect. Because of its size and charge, DNA hardly crosses cell membranes (4). Although viral vec- tors are highly efficient in entering the cell, numer- ous efforts have been done to develop non-viral vectors to ensure safer and easier transfection con- ditions (5). The polyethylenimine (PEI) transfec- tion reagent has already shown promising results for gene delivery of non-viral vectors into cells (6,7). PEI is obtained by acid-catalyzed polymer- ization of aziridine, yielding a highly branched network of molecules with a high cationic charge density (8), which become efficient condensing agents that lead to the formation of small particles (<100 nm) exhibiting good transfection efficiency both in vitro and in various in vivo applications (9). Another advantage of PEI is that it is widely