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

摘要

We describe the use of gene-gun-mediated transfer of luciferase and green fluorescent protein (GFP) reporter genes in zebrafish (Danio rerio). Optimization of DNA transfer parameters indicated highest overall luciferase expression in epidermis and dermis using 1-#mu#m microcarriers and 1 #mu#g of pCMVL plasmid DNA at a delivery pressure of 200 psi. Time course studies revealed luciferase activity peaking at 18 hours and decreasing to 30% of the maximum at day 8 after DNA transfer. onset of reporter gene (GFP) expression was detected at 13 minutes after DNA delivery, and by 65 minutes approximately 100% of the cells in the target area exhibited GFP expression. No germline association or integration events were detected in a screen of approximately 250,000 zebrafish sperm cells by fluorescence in situ hybridizaiton 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 using the biolistic DNA delivery parameters and target tissues described.