Zebrafish Maintenance and Genetic Manipulation

Adult AB zebrafish and larvae were maintained as described previously^{18 (link)}. For live imaging or wounding assay, larvae were anesthetized in E3 containing 0.2 mg/mL Tricaine. To prevent pigment formation, some larvae were maintained in E3 containing 0.2 mM N-phenylthiourea. For morphoino experiments, 2.5–3 dpf larvae were used. For experiments without morpholinos, 3–3.5 dpf larvae were used. Confocal immunofluorescence images were acquired with a confocal microscope (FluoView FV1000, Olympus) using a NA 0.75/20x objective or a NA 1.45/60x oil immersion objective lens. Morpholino oligonucleotides (Gene Tools) in Danieau buffer were injected into 1-cell-stage embryos. Duox splice morpholino (100μM) was injected with p53 morpholino (300 μM) as previously described^{6 (link)}. Lyn splice morpholino lyn MO1 (5′-TCAGACAGCAAATAGTAATCACCTT-3′) (500 μM) or lyn splice morpholino lyn MO2 (5′-GAGTCTGTATTTCAGTACCATTAGC-3′) (500 μM) was used (Danieau buffer as a control). To generate transgenic lines, 1 nL of solution containing 12.5 ng/μL DNA plasmid (tol2-mpx-lyn-GFP or tol2-mpx-lyn C466A-GFP) and 17.5 ng/μL transposase mRNA was injected into the cytoplasm of one-cell stage embryos. Injected embryos were raised to sexual maturity and screened by crossing with wild-type fish to identify founders. F1 embyros were identified by GFP expression and raised to sexual maturity. Experiments were performed on progeny of F1 or F2 outcross with wild-type fish. HEK293 cells were cultured in DME containing 10% FCS. Cells were transfected using Lipofectamine 2000 (Invitrogen).