|Home | About | Journals | Submit | Contact Us | Français|
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.
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)
NOTE: The cylinder will hold up to 3 vials at a time, so you may want to delay fertilization of eggs a few minutes to see if other females already in the tricaine give eggs. This will help speed things up by treating more batches at one time. Also, remember once the press is being used do not put any more fish in the tricaine until the treatment is completed. Allowing fish to stay in the anesthetic too long can kill them. Secondly, if you squeeze fish while the press is being used, and get eggs, they may be too dry to be viable by the time the press can be used again.
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 normal diploids, although some irregular “EP monsters” may occur. Clutches of EP diploids should be largely (70-90%) viable. The presence of haploids in the EP diploid clutch suggests a failure in the EP process (for example, the press failing to hold pressure). The presence of diploids in the haploid clutch suggests incomplete sperm inactivation (for example a failure in the crosslinker).
EP diploids have been used to determine gene-centromere distances1 to map mutants identified in forward genetic screens4. Forward screens of EP diploid embryos are an efficient way to identify genes involved in both early and later developmental processes and are being used today5.
Methods for in vitro fertilization and gynogenetic diploids were developed for zebrafish by Charline Walker in George Streisinger’s lab at the University of Oregon in the 1970s and 1980s. The method described here is unchanged from Charline’s protocols which are available in full in The Zebrafish Book3.