Novel approaches to immortalise salmon fish cells

Aims

This research aims to produce cell lines of many different salmon cell types for use in experiments to understand how viruses cause disease and might be diagnosed or treated, replacing the use of in vivo fish experiments.

Background

With the growth of the fish farming industry, more studies are carried out to understand how disease affects fish in order to find ways to combat them and protect fish welfare and stocks. Such studies are often carried out using groups of live fish experimentally infected in contained aquarium facilities and the effects monitored. Fish cell lines can be used in place of some in vivo experimentation. However, since viruses often cause disease by infecting specific cell types, if these cell types are not available then in vivo experimentation is the only option.

Research details and methods

Primary cells will be dissociated from gill, heart, kidney and spleen dissected from juvenile Atlantic salmon using procedure optimised to maintain cell type diversity. They will be transfected with a panel of plasmids expressing immortalisation factors to become either transiently or permanently immortalised for growth in culture. Green fluorescent protein (GFP) will be incorporated into the plasmid to specifically monitor the growth of the resulting cell lines and allow for isolation and cloning.

Primary cells will be dissociated from gill, heart, kidney and spleen dissected from juvenile Atlantic salmon using procedure optimised to maintain cell type diversity. Cells suspension will be transfected using the Neon electroporation system suitable for transient or stable transfection of fish cells. GFP-expressing plasmid will be used to monitor and optimise transfection parameters for each tissue.

Plasmids producing salmon proto-oncogenes, stem cell inducers and Insulin-Like Growth factor (IGF)-1 are already available in-house and can be used immediately. Coding sequences for anti-apoptotic and Theileria genes are available and will be synthesised commercially and sub-cloned into expression plasmid vectors carrying suitable eukaryotic selection markers. Coding sequences for EGF and TGF-a are available for Zebrafish. The salmon genes will be isolated by a combination of PCR homology and in silico interrogation of the Salmon genome. Where antibody is available, protein expression will be verified by western blot on transiently transfected fish cells. Otherwise qPCR analysis will be used to ensure successful transcription.

If success is limited, double, triple or quadruple transgenic fish cell lines will be attempted sequentially. Four eukaryotic selection markers have been used successfully in fish cells and dual-expression vectors have been engineered giving a potential of eight transgenes being over-expressed in a quadruple transgenic cell line. Double transgenic fish cell lines have been successfully generated.

In every approach, strong proliferation will be the expected phenotype. Inhibition of apoptosis will be measured by reduction in DNA fragmentation and decrease in TUNEL staining. Immortalised cell lines will be characterised by specific staining, morphology and qPCR profiling.

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Pilot study grant

Status:

Closed

Principal investigator

Dr Bertrand Collet

Institution

Marine Scotland Science

Co-Investigator

Dr Catherine Collins

Grant reference number

NC/L000148/1

Award date:

Dec 2013 - Feb 2015

Grant amount

£74,443