Development of microarray techniques for the study of gene expression in the European eel (Anguilla anguilla) during silvering and migration to seawater
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27/06/2008Author
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Abstract
The European eel, Anguilla anguilla, has a complex life-cycle involving
migrations between the Sargasso Sea and the river systems of Europe and
North Africa. The requirement to move across large salinity gradients
presents a significant physiological challenge and the developmental stages
of the eel are closely linked to these migrations. Microarrays were created to
elucidate gene expression changes occurring during;
i. The transition from juvenile yellow to the adult sexually
maturing, migrating silver eel and;
ii. Salinity adaptation during the migration from freshwater to
seawater.
Groups (n = 6) of freshwater-acclimated yellow or silver eels were
transferred to seawater for between 6 hours and 5 months and
complementary control groups were transferred to freshwater. Brain, kidney,
intestine and gill cDNA libraries were constructed using suppression
subtractive hybridisation (SSH) techniques and a novel protocol based on
Invitrogen's Gateway cloning system. The latter technique produced a low
redundancy (~4 %) EST bank with a wide range of insert sizes (0.5 – 10 kb).
Two microarray types were produced; one comprised 5760 clones from the
two brain libraries whilst the other was a multi-tissue microarray incorporating
6144 clones from the SSH libraries. Pooled RNA samples were probed
against the microarrays to highlight differentially expressed genes. Real-time
quantitative PCR (QPCR) was used to validate the observed expression
changes of selected genes in the tissues of individual fish. Following yellow
to silver transformation of freshwater-adapted eels, the expression of tyrosine
3-mono-oxygenase/tryptophan 5-mono-oxygenase activation protein (14-3-3)
and vaccinia related kinase 3 was shown to be consistently elevated.
Prolactin expression increased in the brains of silver eels following two-day
seawater-acclimation but QPCR analysis revealed high variation amongst
freshwater-adapted eels. This is the first eel microarray study and the
expression profiles highlighted herein will provide new avenues for research
into the sexual development and salinity acclimation of A. anguilla
Type
Thesis, PhD Doctor of Philosophy
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Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported
http://creativecommons.org/licenses/by-nc-sa/3.0/
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