Morpholino Mediated Gene Editing: A Gene Knockdown Technique Using Translational Blocking Morpholino Oligonucleotides in Single-Celled Cavefish Eggs

Abstract

Source: Stahl, B. A. et al., Manipulation of Gene Function in Mexican Cavefish. J. Vis. Exp. (2019).

This video describes a gene knockdown technique using translational blocking morpholinos microinjected in the one-cell stage of the cavefish. Morpholinos are DNA oligonucleotides that bind to the complementary sequence of target mRNA and block its translation to a protein. Consequently, morpholinos are used as molecular tools to stop or reduce a gene’s expression transiently.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Morpholino oligo design

NOTE: Sequences for A. mexicanus are available through National Center of Biotechnology Information (NCBI) Gene and NCBI SRA (https://www.ncbi.nlm.nih.gov), as well as from the Ensembl genome browser (https://www.ensembl.org). When designing a morpholino for use in both surface- and cave-dwelling forms, it is critical to identify any genetic variation between the morphs at this stage, so these genetic re....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fish breeding & egg supplies
Fine mesh fish net	Penn Plax	BN4
Fish tank heater	Aqueon	100106108
Egg traps	Custom made	NA	Design and create plastic grate to place at bottom of tank to protect eggs
Glass pipettes	Fisher Scientific	13-678-20C
Pipette bulbs	Fisher Scientific	03-448-21
Agarose	Fisher Scientific	BP160-500
Egg molds	Adaptive Science Tools	TU-1
Morpholino supplies
Control Morpholino	Gene Tools, LLC	Standard control olio
Custom Morpholino	Gene Tools, LLC	NA
Phenol Red	Sigma Aldrich	P0290-100ML
Microinjection supplies
Glass Capillary Tubes	Sutter Instruments	BF100-58-10
Pipette puller	Sutter Instruments	P-97
Picoinjector	Warner Instruments	PLI-100A
Micromanipulator	World Precision Instruments	M3301R
Micromanipulator	Stand World Precision Instruments	M10
Micmanipulator Base	World Precision Instruments	Steel Plate Base, 10 lbs