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Gene Regulation Group
The
Gene Regulation Group (formerly the Molecular Biology Group) at the
National Diagnostics Centre is engaged in research activities
related to the investigation of differential gene expression in a
range of mammalian and fish tissues. Our goal is to improve current
understanding of factors that influence the quality and welfare of
these species from the perspective of both the consumer and the
animal itself. We are also involved in the development of CHO
expression systems for the production of recombinant glycoproteins.
The
group is made up of postgraduate students, research assistants and
postdoctoral researchers. Several of the projects are collaborations
with partners in other European countries including, France, Spain,
Sweden and Britain.
Research in the group focuses on the areas of:
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Aquaculture
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Animal
Biotechnology
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Mammalian
Cell Culture
Contact:
Dr Michael Cairns
Team Leader Gene Regulation Group
National Diagnostics Centre
National University of Ireland, Galway
Ph: 353 91 524411 x2094: 353 91 492094 (direct)
Fax: 353 91 586570
Email: Michael.Cairns@nuigalway.ie
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Aquaculture
There are
currently three funded projects in the Gene Regulation Group related
to Aquaculture.
The overall
aim of the project "A
Functional Genomics Approach to Measuring Stress in Fish Aquaculture"
is to identify in fish candidate genes associated with resistance to
stress conditions and thus provide the physiological and genetic
basis for new marker-assisted selection strategies. The stressful
conditions of intensive aquaculture lead to an overall reduction in
performance, including poor acclimatisation and growth performance,
impaired reproduction and increased susceptibility to disease. The
recent development of genomic tools, particularly microarray
technology, allows systematic gene expression analysis of biological
material and provides an integrated overview of the global response
at the level of gene expression. The present study applies this new
functional genomic approach to examine a complex physiological
problem: analysis of the pattern of gene expression at tissue level
in response to exposure to a stressor.
The project is funded under
EC Framework 5 and has as PIs: Patrick Prunet (Rennes, France), Tom
Pottinger (CEFAS, Windermere), Chris Secombes (Aberdeen), Svante
Winburg (Uppsala, Sweden), Michael Cairns (NUI, Galway) and Andrew
Cossins
(Liverpool). Patrick Prunet is the project coordinator and contact
point. See also the official Stressgenes
web site. NUI, Galway is responsible for preparing SSH cDNA
libraries from liver, pituitary and brain from fish subjected to
confinement stress.
Figure
1
Salmon
salar.
We are also
interested in the isolation of genes involved in the regulation of
the immune system in commercially important salmonid species.
Although numerous cytokines have been isolated from mammals, only a
few cytokines have been identified in teleosts. In this project we
are using the technique of Suppression Subtractive Hybridization
(SSH) to isolate genes differentially expressed in response to
mitogens, such as phytohaemaglutinin (PHA) and lipopolysaccharide
(LPS).
Figure
2
: Printed clones from SSH libraries generated from "confinement
stressed" Rainbow trout (O.mykiss) tissues (brain, liver
& pituitary). The target is RNA extracted from a 2 hour stressed
sample (liver).
In
the project "Regulation of Acute Phase Response (APR)
Proteins in Rainbow trout during Exposure to a Confinement Stressor"
studies into stress in aquaculture are extended. The acute phase
response (APR) is a physiological response of the body to injury,
trauma or infection. The APR involves changes in the hepatic,
neuroendocrine, hematopoietic, musculo-skeletal and immune systems.
Many of the APPs (Acute Phase Proteins) involved in the APR are
synthesised in the liver. Although the APR is typically induced by
injury, trauma and infection in this project we are investigating
whether the APR can also be induced in Rainbow trout by the
confinement / overcrowding stress of the aquaculture environment.
This project is funded by Enterprise Ireland
In
a third project "Molecular Biology of Wild Atlantic
Salmon", NUI, Galway researchers in the National
Diagnostics Centre, the Martin
Ryan Institute and the Department
of Zoology, and their collaborators in the Marine
Institute and University College
Cork, are exploiting functional genomics, proteomics and
population genetics approaches to an investigation of the molecular
genetics of wild Atlantic salmon (Salmo salar). Building on the
existing research interests and expertise of the participants, the
major objective of this programme is to create the first
comprehensive database of gene expression profiles and functional
information associated with embryo development, smoltification and
sexual maturation in wild Atlantic salmon. In a related project we
have investigated thyroid stimulating hormone (TSH) and its role in
smoltification in Atlantic salmon. The thyroid-pituitary axis is of
major importance for the metabolic changes that occur during
smoltification. In order to understand the regulation of hormonal
changes in the Atlantic salmon during smoltification, we have
investigated the cDNA sequences, genomic organisation and promoter
sequence of the thyroid stimulating hormone b
subunit and a
GP subunits. The Wild Salmon project is funded by the Irish Higher
Education Authority.
Figure 3: Returning adult Atlantic
salmon (Salmo salar)
Ø Animal
Biotechnology
There are currently two funded
projects in the Gene Regulation Group related to Animal
Biotechnology.
In the project "New Gene Tools
to Improve Pig Welfare and the Quality of Pork" the overall
aim is to identify genes responsible for variation in pork meat
quality, including the relationship with stress and animal welfare,
in order to provide the basis for the development of new tools to
improve raw material quality. This functional genomics approach
requires phenotypic data that relates to a known genetic structure.
The approach being adopted utilises novel genomics technologies such
as cDNA microarrays and proteomics to analyse gene expression and
gene products in muscle at the time of slaughter in order to relate
this to differences in meat processing and eating quality.This
project is supported by the EC under the Framework 5. For more
information on this project and the partners involved please see qualityporkgenes
Figure 4: Sampling March 2003
A
related project “Analysis
of Gene and Protein Expression for the Measurement of Beef
Quality”
is
funded through the Department of Agriculture, Food and Rural
Development in Ireland by Dr Anne Maria Mullen,
(Coordinator). The
National Food Centre, Teagasc,
Dublin, a collaboration with Dr Torres
Sweeney, Faculty of
Veterinary Medicine, University College Dublin.
Although
many advances have been made in controlling the growth and carcass
composition of animals and the post slaughter conditioning of
carcasses, factors that will ensure a product of consistent quality
are still difficult to define. Meat
quality is an extremely complex set of properties, manifested as
economically important phenotypic traits such as tenderness,
nutrition and water holding capacity.
These are ultimately determined by the interaction of genetic
factors, (through the expression of particular genes leading to the
production of specific gene products (proteins)) with the
environment. This project aims to make progress towards the
identification of genes and proteins (and their expression levels)
that confer the consistently high quality required by both the Irish
and export markets. The importance of these genes and proteins in
determining desirable meat quality will be assessed by analysing
associations between quality traits and the expression of the
identified genes and gene products. More information on meat quality
can be obtained through Teagasc.
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Mammalian
Cell Culture
It is
important in producing recombinant glycoproteins for the human
therapeutic and diagnostic markets that the covalent attachment of
glycan/oligosaccharide chains to the protein is carried out
correctly, as these structures are important for the in vivo
functioning of the glycoprotein. The advantages of recombinant
glycoprotein products over natural products, such as unlimited
source, ease of large-scale production and purification as well as
assurance of product safety and consistency, has led to the rapid
growth in this sector of the biotechnology industry.
For authentic glycosylation, mammalian cell production
systems, such as Chinese Hamster Ovary cells, must be used. The main
objective of this project, which is a collaboration with Prof Terry
Smith in the NCBES is
to characterise novel regulatory elements (promoters, 3'UTRs and
polyadenylation signal sequences) from CHO cells for use in high
efficiency CHO expression systems.
Figure 5: Main room of the Gene Regulation Laboratory
Procedures
used in the Gene Regulation Group
include:
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RNA
isolation and quality determination
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cDNA
and SSH library construction and characterisation
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cDNA
microarray analysis
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Q-PCR
(Real time) to confirm differential expression
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Cell
culture reporter assays and promoter isolation
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