PUBLICATION

Gene transfer and transient expression of transgenes in zebrafish Danio rerio

Authors
Williams, D.W.
ID
ZDB-PUB-021016-90
Date
1997
Source
Ph.D. Thesis : (Thesis)
Registered Authors
Williams, Darren W.
Keywords
none
MeSH Terms
none
PubMed
none
Abstract
The application of transgenic technology to teleost fish has two primary goals; the manipulation of specific traits in commercially important species for economic gain, and the use of small teleost fish, such as the zebrafish (Danio rerio), as a model system to study vertebrate development and gene regulation. To realise each of these goals efficient gene transfer and a reliable means of assaying the functionality of cis-acting regulatory elements are required. The efficiency of gene transfer by egg electroporation was compared with microinjection. Removal of the egg envelope, the chorion, was absolutely necessary to achieve gene transfer by electroporation. The process of dechorionation and the low survival of denuded embryos suggests it is unlikely that the electroporation of dechorionated fish eggs will be adopted as an alternative method of gene transfer until more successful methods of culturing denuded embryos are developed. Using quantitative transient transgene expression assays it was possible to resolve functional differences between cis-acting regulatory elements of different origin, in this case the $beta$-cytoplasmic actin genes of two evolutionarily distant classes of vertebrates. Hybrid 'intron exchange' constructs, demonstrated that cis-regulatory elements residing within carp intron I were necessary for high level expression in fish. Mis-splicing was observed with the equivalent mammalian sequence. High variability in expression levels was found with transient transgene assays, which may seriously influence quantitative comparative analyses. An attempt was made to elucidate the source of this observed variability. High levels of transgene expression were found to occur in the yolk syncytial layer (YSL). The stochastic distribution of reporter gene constructs into YSL or non-YSL compartments, within the embryo, were found to result in strikingly different levels of reporter gene activity. The implications of these findings for the future direction of transgenic research in fish are discussed.
Errata / Notes
Ph.D. Thesis, University of Southampton (United Kingdom)
Genes / Markers
Figures
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping