Novel approaches to immortalise salmon fish cells.
Lead Research Organisation:
Marine Scotland
Department Name: Marine Laboratory
Abstract
Animals are commonly used in biological sciences for both fundamental and applied research. With the growth of the fish farming industry, more and more studies are carried out to understand how disease affects fish in order to find ways to combat them and protect fish welfare.
To gain understanding on how a virus can transmit disease, or how pathogenic it is for the fish experiments are often carried out where a group of fish is experimentally infected in a special containment aquarium facility and the effects monitored. It is sometimes necessary to carry out experiments with fish (in vivo experiments) to answer certain questions but often valuable information can be obtained by performing the experiments instead on immortal fish cells (in vitro experiments). These are cells which divide and grow indefinitely on plastic dishes in a synthetic culture medium containing nutrients and give rise to a cell line, an indefinite source of biological material. The cells divide outwards from a few initial cells to form a layer on the surface of the dish. The cell lines are usually composed on one cell type, e.g. a kidney cell, heart cell or kidney cell. Cell lines can be used to test whether a tissue sample taken from a dead fish contains a virus or not (diagnosing disease), or whether the fish cell can make products to combat the virus (anti-viral response). When the virus is added to the fish cells, it infects the cells and kills them and this forms gaps in the cell layer which are easily seen (cytopathic effect).
Unfortunately, there are many strains of viruses responsible for serious diseases that do not grow very well in the fish cell lines currently available. In addition in fish there is no possibility to control what type of cell will generate a cell line. This is because even if you start with a sample of heart or kidney or gill tissue, these tissues are made up of several cell types and to date, in fish, cell lines are generated "by luck", that is, one of the cell types in the tissue may start to divide and continue to do so, but it may not be the cell type which the virus normally infects. In fact, it is difficult to allocate some of the currently available cell lines to a certain fish tissue.
The present project aims to find new methods to produce fish cell lines from targeted cell types. Cells dissociated from fish organs can be genetically modified to undergo a permanent cell division and survive in synthetic culture medium. These cells would propagate indefinitely and can be used to replace animal experiments by in vitro ones. Several strategies will be tested alone or in combination: immortalisation of cells by factors which have been found to induce cancer cells to divide and grow, by growth factor, by factors which inhibit cell death, by factors produced by parasites known to trigger the proliferation of blood cells. Genetic systems which can produce these factors will be introduced into the initial cells isolated from different tissue types, and the factors will act on the cell to keep it dividing and/or prevent it from dying. The ultimate aim is to reduce the number of live fish needed to investigate and diagnose fish disease and to provide tools for use in improving fish health and welfare.
With increasing pressure on wild fish stocks and an increasing world population, aquaculture has become an important provider of protein for many communities and will most likely continue to grow.
To gain understanding on how a virus can transmit disease, or how pathogenic it is for the fish experiments are often carried out where a group of fish is experimentally infected in a special containment aquarium facility and the effects monitored. It is sometimes necessary to carry out experiments with fish (in vivo experiments) to answer certain questions but often valuable information can be obtained by performing the experiments instead on immortal fish cells (in vitro experiments). These are cells which divide and grow indefinitely on plastic dishes in a synthetic culture medium containing nutrients and give rise to a cell line, an indefinite source of biological material. The cells divide outwards from a few initial cells to form a layer on the surface of the dish. The cell lines are usually composed on one cell type, e.g. a kidney cell, heart cell or kidney cell. Cell lines can be used to test whether a tissue sample taken from a dead fish contains a virus or not (diagnosing disease), or whether the fish cell can make products to combat the virus (anti-viral response). When the virus is added to the fish cells, it infects the cells and kills them and this forms gaps in the cell layer which are easily seen (cytopathic effect).
Unfortunately, there are many strains of viruses responsible for serious diseases that do not grow very well in the fish cell lines currently available. In addition in fish there is no possibility to control what type of cell will generate a cell line. This is because even if you start with a sample of heart or kidney or gill tissue, these tissues are made up of several cell types and to date, in fish, cell lines are generated "by luck", that is, one of the cell types in the tissue may start to divide and continue to do so, but it may not be the cell type which the virus normally infects. In fact, it is difficult to allocate some of the currently available cell lines to a certain fish tissue.
The present project aims to find new methods to produce fish cell lines from targeted cell types. Cells dissociated from fish organs can be genetically modified to undergo a permanent cell division and survive in synthetic culture medium. These cells would propagate indefinitely and can be used to replace animal experiments by in vitro ones. Several strategies will be tested alone or in combination: immortalisation of cells by factors which have been found to induce cancer cells to divide and grow, by growth factor, by factors which inhibit cell death, by factors produced by parasites known to trigger the proliferation of blood cells. Genetic systems which can produce these factors will be introduced into the initial cells isolated from different tissue types, and the factors will act on the cell to keep it dividing and/or prevent it from dying. The ultimate aim is to reduce the number of live fish needed to investigate and diagnose fish disease and to provide tools for use in improving fish health and welfare.
With increasing pressure on wild fish stocks and an increasing world population, aquaculture has become an important provider of protein for many communities and will most likely continue to grow.
Technical Summary
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 8 transgenes being over-expressed in a quadruple transgenic cell line. Double transgenic fish cell lines have been successfully generated (1).
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 (2).
1. Lester K, Urquhart K, Hall M, Gahlawat S, Collet B. 2012. J Virol Meth 182:1-8.
2. Collet B, Collins C. 2009. Vet. Immunol. Immunopathol. 130:92-5
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 8 transgenes being over-expressed in a quadruple transgenic cell line. Double transgenic fish cell lines have been successfully generated (1).
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 (2).
1. Lester K, Urquhart K, Hall M, Gahlawat S, Collet B. 2012. J Virol Meth 182:1-8.
2. Collet B, Collins C. 2009. Vet. Immunol. Immunopathol. 130:92-5
Planned Impact
Currently, there is a lack of suitable cell lines for fish research/diagnostic studies. The development of fish cell lines is extremely empirical and only a few fibroblastic-like immortalised cell lines are available for fish research. As a result, the research community relies on in vivo experiments or has to sacrifice fish to obtain cell types for in vitro studies.
A number of factors have resulted in increasing need for experimental work on fish. Most importantly is the growing importance of aquaculture both in terms of economics and in food security across a wide range of established and new species. This is coupled to the business need and the public demand for better fish welfare. Research has also highlighted the complexity of pathogen virulence, and environmental interaction, which necessitates more refined and extended experimental work to improve control of fish disease and enhance fish welfare. Underlying advances in understanding disease and host response is the rate at which genomic information for commercial farmed fish species is becoming available, with the genome of several species including Atlantic salmon having been sequenced or in the process of being sequenced. As a result and as literature metrics demonstrate, the number of novel fish genes being functionally characterised is increasing. Whole-transcriptome studies highlight the large number of genes associated with important biological functions that remain to be characterised.
The associated cost in number of animals used in experimental procedures to enable this scientific work is following the same increasing trend as that of the fish farming industry. This could be reduced by the provision of a variety of new fish cell types.
In the area of fish biology, there are a number of in vivo experimentations performed by members of the research community with the following objectives:
1. Characterise the role of newly isolated genes in tissues of fish following treatment/pathogen through expression studies.
2. Test the toxicity/pollutants/virulence/effect of a treatment/pathogen/feed
3. Evaluate the susceptibility of different fish species to a pathogen
4. Characterise livestock for resistance to pathogen or environmental parameters.
Looking at examples of virulence studies conducted at Marine Scotland we can forecast a strong reduction in the number of animal used for research. From field case studies, there is evidence that most of the viral agents of serious fish diseases have a strict tissue tropism. During infection, the viraemia phase is followed by active viral replication in a discrete numbers of target organs and cell types. For instance, Salmon Alphavirus, agent of Pancreatic Disease in salmon, replicates mainly in cardiomyocytes. We believe that a good correlation would exist between an in vitro propagation test of SAV on a cardiac cell line and severity of the disease. Infectious Salmon Anaemia virus HPR0 strain is thought to propagate in gill cells, for which no cell lines exist, therefore virus propagation and studies must be done in vivo. In vitro assays could ultimately replace in vivo experimental infections for many studies. These examples can be extended to many aspects of viral disease research and more generally on immunology studies, e.g. provision of leukocyte cell lines
Because of the high phylogenetic conservation of proto-oncogenes, stem cell-inducing transcription factors and growth factors the multiple approaches suggested in this project would be applicable to all fish species.
In addition, this project can potentially generate stable fish cell lines over-expressing salmon IGF-1, TGF-alpha and/or EGF that can be used to produce Foetal Bovine Serum (FBS)-free conditioned tissue culture medium for fish cells. Again, due to the high conservation of these molecules between fish species, this material could be used on any fish species.
A number of factors have resulted in increasing need for experimental work on fish. Most importantly is the growing importance of aquaculture both in terms of economics and in food security across a wide range of established and new species. This is coupled to the business need and the public demand for better fish welfare. Research has also highlighted the complexity of pathogen virulence, and environmental interaction, which necessitates more refined and extended experimental work to improve control of fish disease and enhance fish welfare. Underlying advances in understanding disease and host response is the rate at which genomic information for commercial farmed fish species is becoming available, with the genome of several species including Atlantic salmon having been sequenced or in the process of being sequenced. As a result and as literature metrics demonstrate, the number of novel fish genes being functionally characterised is increasing. Whole-transcriptome studies highlight the large number of genes associated with important biological functions that remain to be characterised.
The associated cost in number of animals used in experimental procedures to enable this scientific work is following the same increasing trend as that of the fish farming industry. This could be reduced by the provision of a variety of new fish cell types.
In the area of fish biology, there are a number of in vivo experimentations performed by members of the research community with the following objectives:
1. Characterise the role of newly isolated genes in tissues of fish following treatment/pathogen through expression studies.
2. Test the toxicity/pollutants/virulence/effect of a treatment/pathogen/feed
3. Evaluate the susceptibility of different fish species to a pathogen
4. Characterise livestock for resistance to pathogen or environmental parameters.
Looking at examples of virulence studies conducted at Marine Scotland we can forecast a strong reduction in the number of animal used for research. From field case studies, there is evidence that most of the viral agents of serious fish diseases have a strict tissue tropism. During infection, the viraemia phase is followed by active viral replication in a discrete numbers of target organs and cell types. For instance, Salmon Alphavirus, agent of Pancreatic Disease in salmon, replicates mainly in cardiomyocytes. We believe that a good correlation would exist between an in vitro propagation test of SAV on a cardiac cell line and severity of the disease. Infectious Salmon Anaemia virus HPR0 strain is thought to propagate in gill cells, for which no cell lines exist, therefore virus propagation and studies must be done in vivo. In vitro assays could ultimately replace in vivo experimental infections for many studies. These examples can be extended to many aspects of viral disease research and more generally on immunology studies, e.g. provision of leukocyte cell lines
Because of the high phylogenetic conservation of proto-oncogenes, stem cell-inducing transcription factors and growth factors the multiple approaches suggested in this project would be applicable to all fish species.
In addition, this project can potentially generate stable fish cell lines over-expressing salmon IGF-1, TGF-alpha and/or EGF that can be used to produce Foetal Bovine Serum (FBS)-free conditioned tissue culture medium for fish cells. Again, due to the high conservation of these molecules between fish species, this material could be used on any fish species.
