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Reverse Genetic Approach to Identify Regulators of Pigmentation using Zebrafish
Published on: March 1, 2022
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Efficient transgenesis mediated by pigmentation rescue in zebrafish.
Itrat Harrold1, Seth Carbonneau2, Bethany M Moore1
1Departments of Pharmacology and Medicine, Cancer Research Center, Section of Hematology and Medical Oncology, Boston University School of Medicine, Boston, MA.
Biotechniques
|January 14, 2016
Summary
This study introduces a new transgenic method in zebrafish using pigmentation rescue for easier identification of transgenic fish. This approach simplifies gene discovery screens by enabling naked-eye sorting, reducing labor and improving efficiency.
Area of Science:
- Genetics
- Developmental Biology
- Aquaculture
Background:
- Zebrafish are crucial for large-scale genetic and drug discovery screens.
- Many transgenic zebrafish lines are heterozygous due to homozygous lethality, requiring progeny sorting.
- Current sorting methods using fluorescence microscopy are labor-intensive and require specialized skills.
Purpose of the Study:
- To develop an efficient transgenic method for zebrafish that allows for naked-eye identification of transgenic individuals.
- To reduce the labor and technical skill required for sorting transgenic zebrafish for screens.
- To improve the efficiency of generating stable transgenic zebrafish lines for large-scale screening.
Main Methods:
- Co-injection of two constructs with the I-SceI meganuclease enzyme into pigmentless nacre mutant zebrafish embryos.
- One construct (I-SceI-mitfa:mitfa-I-SceI) rescues pigmentation, while the other (I-SceI-zpromoter:gene-of-interest-I-SceI) expresses the gene of interest.
- Utilizing pigmentation rescue as a predictor for transgene integration and identification.
Main Results:
- Pigmentation rescue accurately predicted transgene integration in zebrafish.
- The method significantly increased the percentage of founders compared to other transgenic techniques.
- Facilitated accurate naked-eye identification of stable transgenic zebrafish, reducing the need for microscopy and PCR genotyping.
Conclusions:
- This novel transgenic approach simplifies the generation of transgenic zebrafish for large-scale genetic and drug discovery screens.
- The method enhances efficiency by enabling naked-eye sorting, making it ideal for high-throughput screening applications.
- Reduces the overall cost and labor associated with producing transgenic zebrafish lines.
