Gene transfer and transient expression of transgenes in zebrafish Danio rerio
Gene transfer and transient expression of transgenes in zebrafish Danio rerio
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 β-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).
University of Southampton
1997
Williams, Darren William
(1997)
Gene transfer and transient expression of transgenes in zebrafish Danio rerio.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
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 β-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).
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Published date: 1997
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Local EPrints ID: 460282
URI: http://eprints.soton.ac.uk/id/eprint/460282
PURE UUID: 490736ad-a15f-458b-a407-a3cde051c579
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Date deposited: 04 Jul 2022 18:17
Last modified: 04 Jul 2022 18:17
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Author:
Darren William Williams
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