Novel viral delivery systems for genetic engineering of fish cells

The use of viral delivery systems as a tool for foreign gene expression has provided an invaluable resource in a variety of mammalian species. However, although highly efficient in mammals, this tool is not well established in other vertebrate. To this end, we are developing effective viral systems in fish cells, using a wide range of species with commercial value including Carp, Tilapia and Fathead minnow. Additionally, we have developed cell-lines from species with scientific value, such as the turquoise killifish. The Killifish is the shortest-lived vertebrate in captivity, thus providing a unique platform to investigate genetic manipulations in-vivo at an extraordinary speed. We first set up to develop efficient protocols for lentivirus concentration. Importantly, Lentiviruses stably integrate into the host genome, thus enabling long term transgene expression. Using our improved protocols and a lentivirus carrying a GFP reporter, we were able to demonstrate increased infection in several cell lines of killifish, Tilapia, and Fathead Minnow (up to ~ 50%, verified by FACS). Next, in order to increase the viral payload, we turned to baculoviruses, that can deliver large DNA segments into cells (without stable genomic integration). Our preliminary findings demonstrate successful infection efficiency of ~20%. Currently we are engineering viruses with wider host adaptation, which will allow us to expand this toolbox for non-mammalian vertebrates. Our findings will provide an excellent platform for many future applications, including in-vivo genome editing and CRISPR/Cas9 screens for elucidating fish-specific adaptations.