Zebrafish Embryo Microinjection Technique

How To Do A Microinjection of Zebrafish Embryos?

Zebrafish (Danio rerio) embryo microinjection is a powerful technique used in genetic and developmental research. It allows scientists to introduce foreign materials such as DNA, RNA, or CRISPR components directly into the embryo, enabling precise studies of gene function and development. This article provides a detailed guide to setting up for injections, preparing embryos, and performing injections accurately.

1. Preparing for Embryo Injection

Embryo injection requires precision and a well-prepared workspace.

• Essential Equipment for Zebrafish Embryo Injection:
  • Micromanipulator 
  • Microinjector system with fine glass needles
  • Molded agar plates with channels for embryo positioning
  • Dissecting microscope with high magnification for clear visualization
  • Injection material (e.g., plasmid DNA, RNA, or protein solutions)
• Agar Plate Setup:
  • Use an agarose solution to mold plates with channels designed for embryos
  • Each channel should have a vertical side and a 45-degree angled side to support proper positioning during injections
  • Store plates in a clean, sterile environment to prevent contamination

2. Collecting and Loading Embryos

Embryos must be collected and handled carefully to ensure viability during injection.

• Collecting Zebrafish Embryos:
  • Retrieve embryos immediately after spawning using a transfer pipette.
  • Rinse embryos in fish water to remove debris and unviable eggs.
• Loading Zebrafish Embryos onto the Agar Plate:
  • Place embryos into the channels of the agar plate, with the animal pole (the darker region) facing the vertical side of the channel.
  • Align embryos consistently for ease of injection.

3. Preparing the Zebrafish Embryo Microinjection System

• Loading the Needle:
  • Pull fine glass needles using a micropipette puller for optimal injection precision.
  • Backfill the needle with the injection material using a microloader pipette.
  • Calibrate the needle to release the desired volume of material (typically nanoliter-scale).
• Setting Up the Microinjector:
  • Mount the needle onto the microinjector system.
  • Adjust air pressure and injection time to achieve consistent droplet sizes.
  • Test the injection system on a test plate before working with embryos.

4. Performing the Embryo Injection

• Injection Technique:
  • Position the needle at the vertical side of the channel, aimed toward the embryo’s animal pole.
  • Under the microscope, gently penetrate the chorion and inject the material into the yolk or single cell.
• Key Tips for Successful Injection:
  • Use minimal force to avoid damaging the embryo.
  • Inject the material slowly to prevent leakage or overloading.
  • Monitor each embryo post-injection for viability.

5. Post-Injection Care

Injected embryos require proper handling to recover and develop normally.

• Transferring Embryos:
  • Carefully move injected embryos to a clean Petri dish filled with fresh fish water.
• Monitoring Development:
  • Inspect embryos periodically under a microscope to ensure they develop without deformities.
• Temperature and Light Conditions:
  • Maintain embryos at 28°C with a 14-hour light and 10-hour dark cycle to support natural growth.

6. Applications of Zebrafish Embryo Injection

Embryo injections have revolutionized zebrafish research, enabling:

• Gene Editing and Functional Studies:
  • Delivery of CRISPR/Cas9 components for targeted gene knockout or editing.
  • Introduction of mRNA for overexpression studies.
• Transgenesis:
  • Creation of transgenic zebrafish lines by injecting plasmids with fluorescent reporters.
• Drug Screening:
  • Testing the effects of small molecules by injecting compounds directly into embryos.

Conclusion

Zebrafish embryo injection is a cornerstone technique in modern research, allowing unparalleled insights into genetics, development, and disease modeling. By mastering proper preparation, injection techniques, and post-injection care, researchers can maximize the success and reproducibility of their experiments, advancing the field of zebrafish husbandry and genetic engineering.

By Miriam Martínez

Miriam is a Human Biologist with a strong background in neuropharmacology and a passion for bridging science and innovation. After earning a master’s degree in the Pharmaceutical and Biotech Industry, she completed her PhD in Biomedicine at Pompeu Fabra University (Barcelona), where her research focused on the behavioral analysis of animal models for neurophenotypical characterization. Following her doctoral studies, Miriam transitioned into the healthcare marketing and communication sector, where she played a key role in developing impactful marketing strategies and educational campaigns for leading pharmaceutical brands. She now leverages her scientific expertise, strategic thinking, and creative communication skills in her current role at ZeClinics.