Fluorescent Screening of Zebrafish Injected Embryos

Identifying Successfully Injected Embryos With Fluorescence

Fluorescent screening of zebrafish embryos is a powerful method for identifying successfully injected embryos and advancing research objectives such as gene editing, transgenesis, and molecular studies. By using fluorescent markers, researchers can efficiently sort embryos, ensure experimental accuracy, and collect samples for downstream applications like PCR. This guide outlines the key steps involved in fluorescent screening and molecular preparation of zebrafish embryos.

1. Post-Injection Transfer and Cleaning

After microinjection, zebrafish embryos are transferred to clean Petri dishes to ensure their optimal development.

• Initial Transfer:
  • Injected embryos are carefully moved to a new dish filled with fresh fish water.
  • Maintain appropriate density to support growth and reduce competition for oxygen.
• Cleaning and Splitting Embryos:
  • At 4 Hours Post-Injection (hpi):
    • Remove non-viable embryos to prevent contamination.
    • Split viable embryos into separate plates to decrease density, promoting normal development.
  • This meticulous cleaning ensures that the remaining embryos have the best chance to thrive.

2. Screening Injected Embryos with Fluorescent Markers

Fluorescent markers are introduced during microinjection to aid in the selection of successfully modified embryos. While experienced researchers may not need this step, it can be an invaluable tool for those getting started with zebrafish embryo microinjection, helping to build accuracy and confidence in the technique.

• At 24 Hours Post-Fertilization (hpf):
  • Screen for Rhodamine (red fluorescent dye) expression.
  • Rhodamine indicates that the injection material has been successfully delivered into the embryos.
• At 48 Hours Post-Fertilization (hpf):
  • Screen for GFP (Green Fluorescent Protein) expression or other project-specific markers.
  • GFP expression provides confirmation of the incorporation and expression of the injected material, such as DNA or RNA constructs.
• Screening Setup:
  • Use a fluorescence-equipped dissecting microscope to identify fluorescent embryos.
  • Separate fluorescent-positive embryos from non-expressing ones into labeled plates for further processing.

3. Selecting and Processing Zebrafish Embryos for Molecular Assays

After screening for fluorescence—or skipping this step if you are confident in your injection accuracy—embryos are prepared for molecular assays such as PCR.

• Sample Collection:
  • Select fluorescent-positive embryos (e.g., 8–10 embryos per experiment) for further analysis.
  • Ensure embryos are of uniform fluorescence intensity to maintain consistency in experimental results.
• Zebrafish Embryo Lysis Preparation:
  • Euthanize selected embryos using approved protocols.
  • Lyse embryos to extract genetic material:
    • Place embryos in 100 µL of NaOH solution.
    • Heat at 95°C for 45 minutes to break down tissue and release nucleic acids.
    • Neutralize the lysate with 20 µL of TRIS buffer to stabilize the sample.
• Post-Lysis Processing:
  • Store the lysates at -20°C or use immediately for downstream applications like PCR.

4. Applications of Fluorescent Screening and PCR Preparation

Fluorescent screening and molecular assays provide crucial insights for various research projects:

• Gene Editing Validation:
  • Confirm successful delivery and expression of CRISPR/Cas9 or other editing tools.
• Transgenesis Studies:
  • Identify embryos that express introduced transgenes and assess their developmental impact.
• Functional Studies:
  • Use PCR to analyze gene knockouts, knock-ins, or other modifications.
• Drug Screening:
  • Track the effects of injected compounds on gene expression or developmental pathways.

5. Best Practices for Fluorescent Screening of Zebrafish Embryos

• Optimize Marker Selection:
  • Choose fluorescent markers with distinct wavelengths to avoid overlap and ensure clear identification.
• Use Proper Controls:
  • Include non-injected controls to differentiate background fluorescence from true marker expression.
• Maintain Consistency:
  • Ensure uniform injection volumes and conditions across all embryos to improve reproducibility.
• Label Dishes Clearly:
  • Label plates with details such as marker type, time post-injection, and project ID to avoid mix-ups.

Conclusion

Fluorescent screening is a valuable tool in zebrafish research, especially for those new to embryo microinjection or working on projects requiring precise confirmation of injection success. While experienced researchers may choose to bypass this step, leveraging fluorescent markers can streamline the identification of successfully modified embryos, ensuring consistency in downstream applications like PCR. Whether used as a foundational technique or selectively for specific studies, fluorescent screening supports the validation of genetic modifications and advances in developmental biology and drug discovery.

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.