Transgenesis, mutagenesis, knockdown, and genetic colony management

Genetic engineering techniques have rapidly developed since the first large-scale forward genetics screen was developed in 1996, which generated a spectrum of phenotypic zebrafish lines with mutations in many essential genes for normal development. Management of this original collection was relatively straightforward and embryonic analysis was the focus. Adults were heterozygous in their genotype, and phenotypes were primarily within homozygous embryos. Homozygous fish were rarely viable and reporter lines were in their molecular infancy. Genotyping methods were not available since extensive molecular investigation was required to piece together the pathways involved. Mapping and genetic characterization of mutations was propelled forward in the early 2000s by the full sequencing of the Danio rerio genome. This chapter aims to provide a comprehensive overview of the ethical issues in zebrafish (Danio rerio) handling and the most recent practices for the generation and management of zebrafish transgenic, mutant, and mosaic lines. The discussion will take into account recent legislation, international publications, and standard guidelines applied in world reference centers such as the Zebrafish International Resource Center and the European Zebrafish Resource Center, as well as our personal experience in the management of national-level facilities. A set of schemes will be included to provide the reader with an immediate overview of the main operational phases in the different workflows.