|ZFIN ID: ZDB-PUB-030106-17|
A large scale insertional mutagenesis screen in zebrafish - a progress report
|Source:||Proceedings of the EMBO Workshop on Reproduction & Early Development Conference : 62 (Abstract)|
|Registered Authors:||Hopkins, Nancy|
|Keywords:||Fish physiology; Phylogenetics; Genetics; Biological development; Mutagens|
Hopkins, N. (1998) A large scale insertional mutagenesis screen in zebrafish - a progress report. Proceedings of the EMBO Workshop on Reproduction & Early Development Conference. :62.
ABSTRACTOur laboratory showed previously that it is possible to generate proviral insertions in the zebrafish germ line by injecting pseudotyped mouse retroviruses into fish eggs at the 250-2000-cell stages. Using very high titer stocks, it is possible to obtain an average of 10-20 different insertions per founder fish. Founders are highly mosaic since different germ cells are apparently infected independently. Most insertions are transmitted to only a few percent of the F1s. Some germ cells receive multiple proviral integrations and F1 fish with as many as 9 different insertions have been obtained. In a small pilot screen we bred insertions to homozygosity and showed that proviral insertions can integrate into and inactivate genes essntial for embryonic development. The frequency of mutants is low: about 1 mutation per 70-100 insertions. We isolated six recessive embryonic lethal mutations and one dominant adult phenotype. We readily cloned genes that are disrupted in six of the seven mutants, demonstrating the power of insertional mutagenesis in this vertebrate. On the basis of these results, we have begun a large scale insertional mutagenesis screen. It will require the efforts of approximately 20 people for a period of three years. It is hoped that this screen will yield mutations in a significant fraction of the embryonic lethal genes of the fish. The protocol and the current status of this large experiment will be discussed, as well as efforts to increase the frequency of mutagenesis using retroviral vectors.
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