INSERTIONAL MUTAGENESIS AND RAPID CLONING OF ESSENTIAL GENES IN ZEBRAFISH

LARGE-SCALE chemical mutagenesis screens in zebrafish have led to the isolation of thousands of lethal mutations in genes that are essential for embryonic development(1,2). However, the cloning of these mutated genes is difficult at present as it requires positional cloning metho ds, In Drosophila, chemical mutagenesis screens were complemented with P-element insertional mutagenesis which facilitated the cloning of ma ny genes that had been identified by chemical lesions(3,4). To facilit ate the cloning of vertebrate genes that are important during embryoge nesis, we have developed an insertional mutagenesis strategy in zebraf ish using a retroviral vector, Here, in a pilot screen of 217 proviral insertions, we obtained three insertional mutants with embryonic leth al phenotypes, and identified two of the disrupted genes, One of these , no arches, is essential for normal pharyngeal arch development, and is homologous to the recently characterized Drosophila zinc-finger gen e, clipper, which encodes a novel type of ribonuclease(5). As it is ea sy to generate tens to hundreds of thousands of proviral transgenes in zebrafish(6), it should now be possible to use this screening method to mutate and then rapidly clone a large number of genes affecting ver tebrate developmental and cellular processes.