Making Gynogenetic Diploid Zebrafish by Early Pressure

Summary

This is a method for generating gynogenetic diploid zebrafish embryos (embryos whose only genetic contribution comes from the mother) by blocking the second meiotic division immediately after fertilization with ultraviolet light-inactivated sperm. EP embryos are not fully homozygous due to recombination during the first meiotic division, however they are homozygous at all loci that have not been separated from their centromere by recombination.

Abstract

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from heterozygous females allow F1 mutagenized females to be screened directly for deleterious mutations in an accelerated forward genetic screen. Streisinger et al.^1,2 described methods for making gynogenetic (homozygous) diploid zebrafish by activating zebrafish eggs with ultraviolet light-inactivated sperm and preventing either the second meiotic or the first zygotic cell division using physical treatments (heat or pressure) that deploymerize microtubules. The "early pressure" (EP) method blocks the meiosis II, which occurs shortly after fertilization. The EP method produces a high percentage of viable embryos that can develop to fertile adults of either sex. The method generates embryos that are homozygous at all loci except those that were separated from their centromere by recombination during meiosis I. Homozygous mutations are detected in EP clutches at between 50% for centromeric loci and less than 1% for telomeric loci. This method is reproduced verbatim from the Zebrafish Book³.

Protocol

Overview of early pressure

Note: recipes for many of the reagents used in this protocol such as tricaine, fish water and Hanks saline are available online in chapter 10 of the Zebrafish Book³ (http://zfin.org/zf_info/zfbook/zfbk.html)

1. Produce embryos by in vitro fertilization using UV inactivated sperm.
2. Immediately transfer the fertilized eggs to a glass vial (with a plastic snap cap) filled to the brim with egg water. Cover the open end of the vial with a thin sheet of latex rubber and snap the top (into which a hole has been cut) onto the vial over t....

Discussion

It is important to make sure that embryos produced by this method are true gynogenetic diploids. As controls, generate a clutch of normal diploid eggs (by keeping aside some of the sperm without UV-inactivation) and a clutch of haploid embryos (by keeping aside a clutch of eggs fertilized with UV sperm without going through the EP procedure). At 1 day post fertilization haploid embryos have a short body axis, irregular brain and somite morphology. Haploids are not viable after 2-3 days. EP diploids should look like norma.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tricaine	Sigma-Aldrich
Watch glass
French Press
Pressure cylinder
Instant ocean
Pasteur pipettes			Cut off the narrow end, fire polish
UV cross-linker
microcapillaries