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

Overview

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 regions can be avoided as targets for morpholinos. Any polymorphic variation within a morpholino target site can lead to ineffective binding. The design is similar to other fish systems, such as zebrafish, and has previously been shown to work effectively in A. mexicanus.

1. Design of translation-blocking morpholinos
   NOTE: Translation-blocking morpholinos block translation by binding to the endogenous start site and impede translational machinery from binding the mRNA sequence through steric hindrance.
   1. Identify the coding region of the target gene starting with the ATG start site.
   2. Record the first 25 base pairs of the target sequence by copy-and-pasting the sequence in a text editor or lab notebook.
   3. Using either online software (e.g., http://reverse-complement.com) or manual translation, generate the reverse complement of the target sequence. Save the resulting reverse complement in a text editor or lab notebook.
   4. Order a morpholino with the reverse complement sequence from a company that generates morpholino oligonucleotides. See Table of Materials for companies.

2. Microinjections

1. Preparation of general tools for injections
   NOTE: The procedures in this section have been described in detail elsewhere, and an overview with minor modifications is presented here.
   1. Generate injection plates by pouring warm 3% agarose dissolved in fish system water into a 100 mL Petri dish. Carefully place an egg injection mold in the freshly poured agarose to make wells for the fish eggs. Place the side of the mold in agar at a 45° angle and then, slowly lower it into the agarose; slowly lowering the mold at an angle avoids air getting trapped underneath the mold. Gently remove the mold once the agarose is solidified. The plates can be stored, sealed, at 4 °C for up to 1 week.
2. Collection of single-cell stage eggs
   1. The night in which breeding is expected, examine the tanks every 15–30 min and monitor for eggs at the bottom of the tank. Eggs appear translucent, measuring approximately 1 mm in diameter.
   2. Use a fine mesh fish net to collect eggs and transfer them to a glass bowl filled with fresh fish system water. Examine the eggs under a microscope to confirm that the eggs are at the one-cell stage.
   3. Using glass pipettes, transfer single-cell eggs to the injection plates. Glass pipettes are required at this stage as the eggs will stick to plastic.
   4. Using a pipette, carefully release the eggs into the wells of the agarose injection plate from section 2.1. Fill the rows of the prewarmed (at room temperature) injection plate with the maximum number of eggs (30–40 per row, and up to five rows). Full rows help keep the eggs from moving during injections. Keep the eggs hydrated on the injection plate with a small amount of fish system water until the performance of the injections.
3. Pico-injection setup and general injection optimization guidelines
   1. Backfill the injection needles using either gel-loading pipette tips that fit inside the capillary or using standard micropipette tips and adding a 2–4 μL bolus to the end. Once the needle is filled, use forceps to trim the excess length from the injection needle.
   2. Perform microinjections using a needle mounted in a micromanipulator, connected to a picolitre microinjector.
   3. Set the injection time to 0.03 s and the pressure out at ~0.0 psi. The injection pressure will vary accordingly with minor differences between needles, so optimize to achieve a ~1.0 nL injection bolus.
      NOTE: The injection pressure is often in the range of ~10–30 psi.
   4. Standardize the injection bolus by injecting into mineral oil and measuring the bolus size with a slide micrometer to achieve a ~1–1.5 nL injection volume. Adjust the injection pressure (psi) to increase or decrease the bolus volume.
   5. Draw water off of the very top of the eggs using a lab tissue.
      NOTE: The Astyanax egg chorion is slightly tougher to penetrate than zebrafish eggs. We find that drawing the water off of the top of the eggs helps facilitate needle penetration into the egg. Plate optimization can allow for ~200 eggs on a single plate.
   6. Use the micromanipulator to penetrate each egg with the needle and inject it directly into the yolk. Once positioned in the yolk, inject the egg by pressing the inject button or injection foot pedal.
      NOTE: A full plate can be injected within ~15 min. The single-cell stage lasts for ~40 min.
4. Injection of morpholinos
   NOTE: The amount of morpholino necessary for knockdown without causing toxicity will need to be optimized per gene target; however, a concentration of 400 pg is a good place to start.
   1. Prepare morpholino so that 400 pg of morpholino will be injected per egg. Thaw morpholino on ice. The injection solution is comprised of morpholino (at the desired concentration), RNase-free H2O or Danieau’s solution, and phenol red (10% of the final volume). For an example, see Table 1.
   2. Inject 1 nL per embryo.

Materials

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