The protocol describes targeted knockdown of gene expression in zebrafish by morpholino injection in the tail.
Protocol
1. Morpholino Injection The day prior to injecting the morpholino, make an injection plate (Figure 1). Make sure to cut out a notch at one end of the well, which helps stabilize the fish for the microinjection procedure. Dilute the fluorescein-tagged morpholino to the proper concentration (recommend starting with 1.2 mM) and place in a 65 °C water bath, for 5 minutes. Load the needle with the morpholino (note: depending on whether you…
Representative Results
Figure 1. A. Schematic of the injection plate, which is made from agarose and contains a small well to hold the fish during microinjection of the morpholino. Figure 2. Schematic of morpholino microinjection. A. Place the fish in the dish with the he…
Materials
Morpholino
GeneTools, LLC
Morpholino should be custom designed to your protein of interest
Micro-injection pump
World Precision Instruments, Inc.
PV830
Pneumatic PicoPump Many different microinjection systems could be used
Micro-manipulator
World Precision Instruments, Inc.
MMJR
Right-handed (MMJL for lefthanded)
Micro-injection needles, 1.0 mm outside diameter
World Precision Instruments, Inc.
1B100F-4
These are borosilicate glass capillaries, pulled into a needle
Needle holder
World Precision Instruments, Inc.
5430-ALL
Pico Nozzle Kit; make sure to inset the 1.0 mm pipette gasket
Needle puller
Sutter Instrument Co.
P-97
Other micropipette/needle pullers should also work
Microscope
Leica, Nikon, Zeiss
Number varies depending on the manufacturer
Any stereomicroscope with 20X optics and the ability to work with micromanipulators
2-Phenoxyethanol
Sigma-Aldrich
77861-1L
Anesthesia; dilute 1:1000 in fish system water for procedure, 1:500 for euthanasia
Morpholino Injection: A Method to Knockdown Target Gene Expression to Study Function in Zebrafish. J. Vis. Exp. (Pending Publication), e20182, doi: (2023).