Effective targeted gene knockdown using morpholino antisense oligos in early development of zebrafish

Authors：

Category

Glycogene transgenic animals

Protocol Name

Authors

Yoshimura, Yukihiro *
Faculty of Health and Welfare Science, Okayama Prefectural University

Ito, Makoto
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University

*To whom correspondence should be addressed.

KeyWords

Gene knockdown Morpholino antisense oligonucleotide Zebrafish GlcT-1 Microinjection

Reagents

●	Morpholino oligonucleotide (GeenTools) MO is dissolved in distilled water at 1–4 mM (stock solution). Before the phenotype is analyzed, the working concentration of MOs and knockdown efficiency must be checked by RT-PCR (splicing-blocker), Western blotting (splicing-, and translation-blocker), or enzymatic assay (if the product of the target gene has enzymatic activity).
●	E3 embryo medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, 0.33 mM MgSO₄, and 10^-5% methylene blue)

Instruments

●	Embryos The night before the injection, place one male and one female separately into a mating box in which each fish is separated by a divider. On the morning of the injection, the fish are put together and usually will mate within 10 min. For efficient knockdown, the injection into embryos should be done at the one- to two-cell stage (approximately 60 min after mating).
●	Microinjection plate As zebrafish eggs are fairly buoyant and move easily during injection, it is recommended to use a microinjection plate in which eggs are kept at the correct position for injection. Narrow slope slots (2–3 mm wide, 1–2 mm depth, slope at 30–45°) are made on an acryl plate to use it as a microinjection plate. Alternatively, the microinjection plate can be replaced with 1.5% agarose.
●	Microinjection needle To prepare microinjection needles, pull glass capillaries (e.g. GD-1, Narishige Co., Ltd., Tokyo, Japan) on a needle puller (e.g. PC-10, Narishige Co., Ltd.). The diameter of the needle should be thick enough to allow easy penetration of the chorion, however too much diameter may damage the embryo. As the injection should be done within 60 min in after mating, it is recommended that the needles be prepared before starting the experiment.
●	Microinjection apparatus A typical microinjection set-up consists of a stereomicroscope, a micromanipulator (e.g. M-152, Narishige Co., Ltd.), and a pneumatic microinjector (e.g. IM-30, Narishige Co., Ltd.)
●	Forceps (No.5, INOX, Muromachi Kikai Co., Ltd., Tokyo, Japan)
●	Microloader pipet tips (Eppendorf AG, Hamburg, Germany)

Methods

Effective targeted gene knockdown using morpholino antisense oligos in early development of zebrafish

1)	Attach the microinjection needle to the manipulator before collecting eggs. Fill the needle with a suitable MO solution using a microloader pipet tip (see Comment). Attach the needle with the MO solution to the micromanipulator.	Comment 1

2)	Collect the eggs into a 10-cm Petri dish filled with E3 medium or water for culture.	Comment 0

3)	Transfer and arrange the eggs within the slots of the microinjection plate (see Comment). Place the plate under a stereomicroscope and make sure that the embryos are at the one- to two-cell stage.	Comment 1

4)	Break off the tip of the needle with fine forceps under a stereomicroscope. Use the manipulator to bring the needle tip close to the embryo. Discharge the pressure using the foot switch (e.g. 25 psi, 100 msec) and estimate the volume (diameter) of a droplet from the diameter of the embryo (see Comment ). Adjust the volume by changing the discharge time (see* Comment **).	Comment 1

5)	Drive the tip of the needle into the yolk of the embryo through the chorion under the control of the micromanipulator. Discharge the solution into the yolk of the embryo (see Comment).	Comment 1

6)	Withdraw the tip from the embryo by lifting the needle slowly (see Comment).	Comment 1

7)	Move the plate slightly to locate the next embryo, and inject the solution.	Comment 0

8)	Tilt the microinjection plate and add E3 medium to the slots to slide the embryos down to a dish filled with E3 medium.	Comment 0

Incubate the dish at 28.5°C until the time of observation or harvest (see Comment).

Comment 1

Figure & Legends

Fig. 1. Structure and mechanism of MOs

a) Structure of MO with target RNA. Note the six-membered morpholino ring (green) and the non-ionic phosphodiemidate link between the two rings (red). b) Inhibition of translation by a translation-inhibitor MO. The MO targets the 5’ of the start codon and inhibits progression of the initiation complex. c-e) Schematic diagram of actions of the splicing- blocker MO.

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Date of registration：2015-11-16 14:10:49