Materials

1. Breeding pairs of adult zebrafish are maintained at 28.5°C with a 14-h light and 10-h dark cycle. Embryos are obtained by natural spawning or by in vitro fertilization (refer to the Zebrafish Book [5] for the method).

2. The microinjection system consists of a stereo microscope, a three-dimensional micromanipulator, and an automatic pressure microinjector.

3. An injection chamber is made by placing a glass microscope slide into a 85-mm diameter disposable plastic Petri dish. Flood the injection chamber with embryo medium (5), and allow a thin film of liquid to form between the slide and the dish. Remove the excess liquid using a glass pipet.

4. Microinjection pipets are prepared as follows: Pull fine glass capillaries with inner filament (Clark Electromedical Instruments, Reading, UK, 1.0-mm od, and 0.58-mm id) with a micropipet puller (Model P-87, Sutter Instrument Co. Novato, CA, or any equivalent). The tip of the micropipet should be about 0.05-mm. If the tip is too thin, it will lack the tensile strength to penetrate the chorion. Thicker

From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ

pipets do not easily withdraw from the chorion without dragging the embryo out of the chamber after injection. Just before microinjection, break the micropipet tip under a microscope with a pair of blunt forceps to produce a sharp end.

5. Molecular biology grade reagents should be used to prepare the solutions, DNA, and RNA samples.

0 0

Post a comment