Undertaking a successful gynogenetic haploid screen in zebrafish

Area of Science:

• Genetics
• Developmental Biology
• Aquatic Toxicology

Background:

• Chemical mutagenesis is essential for generating genetic diversity in model organisms.
• N-ethyl-N-nitrosourea (ENU) is a potent mutagen widely used for dense mutagenesis screens in zebrafish.
• Efficient screening methods are crucial for maximizing the yield of heritable mutations.

Purpose of the Study:

• To describe a streamlined method for pre-meiotic chemical mutagenesis in zebrafish.
• To detail the production of haploid embryos for efficient screening of induced mutations.
• To present an alternative to classical F2 screens that conserves resources.

Main Methods:

• Pre-meiotic sperm mutagenesis using N-ethyl-N-nitrosourea (ENU).
• Crossing mutagenized males with wild-type females to generate heterozygous F1 offspring.
• Production of haploid embryos via in vitro fertilization (IVF) with ultraviolet (UV)-irradiated sperm from F1 females.

Main Results:

• The described method allows for direct screening of mutations in the F1 generation.
• This approach significantly reduces the number of fish and aquarium space required compared to F2 screens.
• The protocol is feasible for smaller research laboratories.

Conclusions:

• Pre-meiotic ENU mutagenesis combined with haploid embryo screening offers an efficient strategy for zebrafish genetic screens.
• This method optimizes resource utilization, making dense mutagenesis more accessible.
• Detailed protocols for haploid embryo production facilitate implementation.