A User-Friendly Protocol for Microinjection into Teleost Embryos to Study Gene Function

Area of Science:

• Developmental Biology
• Functional Genomics
• Teleost Model Organisms

Background:

• Zebrafish and medaka are key teleost models in biological research.
• High-quality, reproducible microinjections are crucial for genetic studies.
• Existing protocols lack unification and optimization for both species.

Purpose of the Study:

• To develop a comprehensive and optimized protocol for microinjection in zebrafish and medaka.
• To enable efficient, precise, and scalable genetic manipulation in these model organisms.
• To support advanced functional studies in developmental biology and disease modeling.

Main Methods:

• Controlled photoperiod breeding tank setup for maximized egg yield and minimized contamination.
• Detailed procedures for sex identification, pair selection, and enhanced egg collection.
• Standardized methods for injection gel bed preparation, needle pulling, and pico-liter injector calibration for nanoliter-scale injections.
• Application for gene knockdown (morpholino), gene knockout (CRISPR-Cas9), and mRNA overexpression.

Main Results:

• Achieved consistent nanoliter-scale injections with minimal variability.
• Demonstrated successful gene manipulation via morpholino, CRISPR-Cas9, and mRNA overexpression.
• Observed consistent phenotypic outcomes between morpholino and CRISPR-Cas9 approaches.
• Reported 97.7% phenotype penetrance in CRISPR-Cas9 knockouts with a triple validation approach.

Conclusions:

• The developed protocol is easy, comprehensive, and enables efficient, precise, and scalable genetic manipulation in zebrafish and medaka embryos.
• This unified approach supports advanced functional studies and disease modeling.
• The triple validation method confirms gene function across multiple techniques, enhancing reliability.