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Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish
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Protocol for tissue-specific mutagenesis with fluorescent labeling in zebrafish.

Juanjuan Luo1, Chunjiao Lu1, Xiaojun Yang1

  • 1Engineering Research Center of Key Technique for Biotherapy of Guangdong Province, Shantou University Medical College, Shantou, China.

STAR Protocols
|July 26, 2024
PubMed
Summary

This study introduces a new protocol for creating tissue-specific mutations in zebrafish. This method enables the generation of stable knockout lines for studying mosaic loss-of-function phenotypes.

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Area of Science:

  • Genetics and Genomics
  • Developmental Biology
  • Zebrafish Models

Background:

  • Understanding gene function often requires targeted gene knockout.
  • Generating tissue-specific mutations is crucial for studying genes with complex roles in development and disease.
  • Zebrafish are a powerful model organism for genetic studies due to their rapid development and optical transparency.

Purpose of the Study:

  • To present a novel protocol for generating stable, tissue-specific mutagenesis in zebrafish.
  • To enable the analysis of mosaic loss-of-function phenotypes in a targeted manner.
  • To provide a detailed method for creating and characterizing tissue-specific knockout zebrafish lines.

Main Methods:

  • Development of a Tol2 transposase donor vector with a U6 promoter for single-guide RNA (sgRNA) and Cas9 expression.
Keywords:
CRISPRDevelopmental biologyGenetics

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  • Utilizing a tissue-specific promoter to drive Cas9 and sgRNA transcription.
  • Establishment, identification, and phenotypic analysis of stable, tissue-specific mutagenesis zebrafish lines.

    • Main Results:

    • Successful generation of a protocol for tissue-specific mutagenesis in zebrafish.
    • Demonstration of the ability to create stable zebrafish lines with targeted gene knockouts.
    • Validation of the protocol for analyzing mosaic loss-of-function phenotypes.

    Conclusions:

    • The presented protocol offers a robust method for generating tissue-specific knockout zebrafish.
    • This technique is valuable for dissecting gene function in a spatially and temporally controlled manner.
    • The protocol facilitates the study of complex genetic interactions and developmental processes in zebrafish.