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Protocol for conditional mutagenesis in zebrafish germ cells using Tol2 transposon and a CRISPR-Cas9-based plasmid
Yixuan Hu1, Yutao Chen1, Yu Zhang1
1College of Life Sciences, Northwest Normal University, Lanzhou 730070, China.
STAR Protocols
|December 22, 2024
Summary
This study introduces a simple and efficient protocol for conditional mutagenesis in zebrafish germ cells. The method utilizes Tol2 transposon and CRISPR-Cas9 technology for straightforward gene disruption in zebrafish.
Area of Science:
- Genetics
- Molecular Biology
- Zebrafish Research
Background:
- Conditional mutagenesis is crucial for studying gene function.
- Zebrafish are a valuable model organism for genetic studies.
- Existing methods for germline mutagenesis can be complex or time-consuming.
Purpose of the Study:
- To present a simplified protocol for conditional mutagenesis in zebrafish germ cells.
- To enable efficient gene disruption using Tol2 transposon and CRISPR-Cas9.
- To facilitate ease of execution and time efficiency in genetic manipulation.
Main Methods:
- Development of a CRISPR-Cas9-based plasmid system.
- Integration with the Tol2 transposon for germline transmission.
- Zebrafish embryo microinjection and screening for phenotypic markers (green fluorescence in the heart).
Main Results:
- Successful implementation of a conditional mutagenesis protocol.
- Demonstration of gene disruption in zebrafish germ cells.
- Protocol is characterized as simple, time-efficient, and multifunctional.
Conclusions:
- The presented protocol offers a user-friendly approach for conditional germline mutagenesis in zebrafish.
- This method simplifies the process of gene disruption, accelerating genetic research in zebrafish.
- The protocol's efficiency and multifunctionality make it a valuable tool for the scientific community.
