Caudal Fin Regeneration in Zebrafish - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 302

Figure 22.3 Larval caudal fin amputation rig. (a) Photograph depicting the microamputation rig.
(b) Embryos at 2 or 3dpf are placed on an agar plate under a microscope and caudal fin tissue is amputated
distal to the notochord with a diamond blade.

embryos (2.5-4hpf) into a 60mm glass Petri dish with 25mL of fish water. Add 50 L

of 50mg/mL pronase to the center of the dish and gently and continuously swirl the
solution for approximately 7min. During this period, routinely observe the embryos
under a microscope to identify embryos without chorions, chorion remnants in the
solution, or deflated chorions. When the above conditions are observed or 7min have
passed, remove the pronase solution by diluting the solution with fresh fish water,
swirling, and slowly decanting. Repeat this step for 10min (approximately 10 washes)
in order to dilute the pronase solution and remove displaced chorions. Allowembryos to
recover at 28 C until a minimum of 6hpf. Manual dechorionation is an appropriate
alternative to chemical dechorionation when using lower numbers of embryos.

m

22.6 STRATEGIES USED TO MANIPULATE GENE
FUNCTION DURING FIN REGENERATION

22.6.1 Mutations

Chemical mutagenesis using N -ethyl- N -nitrosourea (ENU) is a commonly used
chemical mutagen in zebrafish. Mutagenesis of spermatogonia introduces point
mutations into the genome that result in both gain-of-function and loss-of-function
mutants. Mutagenized populations are generally screened for developmental mutants