ZFIN ID: ZDB-PUB-990414-25
Insertional mutagenesis in zebrafish using a pseudotyped retroviral vector
Gaiano, N.R.
Date: 1997
Source: Ph.D. Thesis : (Thesis)
Registered Authors: Gaiano, Nicholas
Keywords: none
MeSH Terms: none
PubMed: none
Large-scale chemical mutagenesis screens have recently been performed in zebrafish and have isolated thousands of mutations affecting processes ranging from epiboly and gastrulation to organogenesis and behavior. However, because these lesions are point mutations, the cloning of the mutated genes is likely to be difficult. With this issue in mind, we undertook the development of an insertional mutagenesis methodology for use in zebrafish. Insertional mutagenesis has been shown to be an effective way to mutate and rapidly clone genes in a wide variety of organisms including D. melanogaster, C. elegans, and mice. First, a pseudotyped retroviral vector, composed of a nucleocapsid based on the Moloney murine leukemia virus (MoMLV) and an envelope derived from the vesicular stomatitis virus (VSV), was injected into blastula-stage zebrafish embryos and shown to be capable of generating transgenic offspring. In an effort to improve upon the obtained transgenic frequency, two new MLV/VSV vectors, prepared to titers roughly 100-fold higher than that of the previously used virus, were injected into embryos. For one of these, injected fish transmitted, on average, 11 different insertions to 30% of their F1 progeny. At this frequency, it is feasible for a single lab to generate tens to hundreds of thousands of proviral insertions. In a pilot insertional mutagenesis screen of 217 insertions, 3 insertional mutations were isolated, and in two cases the disrupted gene was rapidly cloned. One of these encodes a putative endoribonuclease essential for normal pharyngeal arch development, and the other encodes a novel protein necessary for embryonic maturation. As it is easy to generate many thousands of proviral transgenes in zebrafish, it should now be possible to use this screening method to mutate and then rapidly clone hundreds of gene affecting vertebrate developmental and cellular processes. (Copies available exclusively from MIT Libraries, Rm. 14-0551, Cambridge, MA 02139-4307. Ph. 617-253-5668; Fax 617-253-1690.)
Ph.D. Thesis, Massachusetts Institute of Technology, Department of Biology