Gene-gun-mediated transfer of reporter genes to somatic zebrafish (Danio rerio) tissues

We describe the use of gene-gun-mediated transfer of luciferase and green f luorescent protein (GFP) reporter genes in zebrafish (Danio rerio). Optimiz ation of DNA transfer parameters indicated highest overall luciferase expre ssion in epidermis and dermis using 1-mu m microcarriers and 1 mu g of pCMV L plasmid DNA at a delivery pressure of 200 psi. Time course studies reveal ed luciferase activity peaking at 18 hours and decreasing to 30% of the max imum at day 8 after DNA transfer. Onset of reporter gene (GFP) expression w as detected at 13 minutes after DNA delivery, and by 65 minutes approximate ly 100% of the cells in the target area exhibited GFP expression. No germli ne association or integration events were detected in a screen of approxima tely 250,000 zebrafish sperm cells by fluorescence in situ hybridization at 15 or 30 days after delivery of 1 mu g of pCMVL DNA, suggesting incidental male germline integration should not be considered as a risk factor when u sing the biolistic DNA delivery parameters and target tissues described.