Morpholino Injection: A Method to Knockdown Target Gene Expression to Study Function in Zebrafish
Published: April 30, 2023
Abstract
Source: Hyde D.R., et al. In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin. J. Vis. Exp. (2012)
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 |
Citar este artigo
Morpholino Injection: A Method to Knockdown Target Gene Expression to Study Function in Zebrafish. J. Vis. Exp. (Pending Publication), e20182, doi: (2023).