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. However, the cloning of these mutated genes is difficult at present as it requires positional cloning methods. In Drosophila, chemical mutagenesis screens were complemented with P-element insertional mutagenesis which facilitated the cloning of many genes that had been identified by chemical lesions. To facilitate the cloning of vertebrate genes that are important during embryogenesis, we have developed an insertional mutagenesis strategy in zebrafish using a retroviral vector. Here, in a pilot screen of 217 proviral insertions, we obtained three insertional mutants with embryonic lethal 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 gene, clipper, which encodes a novel type of ribonuclease. As it is easy to generate tens to hundreds of thousands of proviral transgenes in zebrafish, it should now be possible to use this screening method to mutate and then rapidly clone a large number of genes affecting vertebrate developmental and cellular processes.
Gaiano
N
N
Center for Cancer Research, Massachusetts Institute of Technology, Cambridge 02139, USA.
Amsterdam
A
A
Kawakami
K
K
Allende
M
M
Becker
T
T
Hopkins
N
N
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004625:Embryo, Nonmammalian / Q000002:abnormalities; D047108:Embryonic Development; D005822:Genetic Vectors; D006801:Humans; D009052:Leukemia Virus, Murine / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010614:Pharynx / Q000196:embryology; D011506:Proteins / Q000235:genetics*; D011533:Proviruses / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics; D015027:Zebrafish / Q000196:embryology / Q000235:genetics*; D029961:Zebrafish Proteins; D016335:Zinc Fingers / Q000235:genetics
D011506:Proteins; D029961:Zebrafish Proteins; C101996:cpsf4 protein, zebrafish