Undertaking a successful gynogenetic haploid screen in zebrafish.
Chemical mutagenesis using N-ethyl-N-nitrosourea is the current method of choice for dense mutagenesis in zebrafish. Methods are available for both pre-meiotic and post-meiotic sperm mutagenesis; in this chapter, pre-meiotic mutagenesis is described. Mutated males are crossed with untreated females to create an F1 generation that is heterozygous for the mutations. The F1 females can be screened directly by making haploid embryos using in vitro fertilization (IVF) with ultraviolet (UV)-irradiated sperm. This approach requires substantially fewer fish and less aquarium space than the classical F2 generation screen and is feasible for a small research group. Production of haploid embryos is described in detail.
The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3050, Australia. jlayton@wehi.edu.au
Judith E
JE
Layton
D000477:Alkylating Agents / Q000008:administration & dosage; D000818:Animals; D004247:DNA / Q000187:drug effects / Q000528:radiation effects; D005038:Ethylnitrosourea / Q000008:administration & dosage; D005260:Female; D005307:Fertilization in Vitro; D005818:Genetic Engineering / Q000379:methods; D005820:Genetic Testing / Q000379:methods*; D006238:Haploidy; D006579:Heterozygote; D008297:Male; D016296:Mutagenesis; D013094:Spermatozoa / Q000187:drug effects / Q000528:radiation effects; D014466:Ultraviolet Rays; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*
D000477:Alkylating Agents; D004247:DNA; D005038:Ethylnitrosourea