In vivo functionality of specific immune genes - by CRISPR gene knockouts and disease challenge
Main goal:
To test the disease-protective function of specific innate immune genes, by knocking them out in fish eggs using CRISPR and testing by disease challenge when the knock-out fish have reached the fry stage.
The hypothesis is that immune gene knock-out fish are more susceptible to experimental challenge with salmonid alphavirus subtype 3 (SAV3), and that the increased susceptibility will present itself as increased viral load, hastened infection kinetics and increased severity of resulting clinical disease.
The application includes ten (10) separate challenge trials performed in context of the NFR project TUNESAL (NFR15676), over a period of 4 years.
Each trial will compare how specific gene knockout salmon groups and wildtype salmon fry will respond to challenge with wildtype SAV3 isolate and/or attenuated SAV constructs that we have produced.
Harmfulness – The experiments will be severe – SAV3 will cause disease and mortality in fry.
Animals - The experiment will involve 10 times 680, i.e. 6800, Atlantic salmon fry (Salmo salar).
3R - The challenge trials will focus on the early infection kinectics, allowing them to be ended/terminated before SAV3-caused mortality will develop beyond the the sporadic. We will employ a salmon fry challenge model that we have developed in recent years, which we find more standardized than many other existing fish models. This constitutes a refinement of methodology, that will reduce the number of fish needed in each specific experiment.
To test the disease-protective function of specific innate immune genes, by knocking them out in fish eggs using CRISPR and testing by disease challenge when the knock-out fish have reached the fry stage.
The hypothesis is that immune gene knock-out fish are more susceptible to experimental challenge with salmonid alphavirus subtype 3 (SAV3), and that the increased susceptibility will present itself as increased viral load, hastened infection kinetics and increased severity of resulting clinical disease.
The application includes ten (10) separate challenge trials performed in context of the NFR project TUNESAL (NFR15676), over a period of 4 years.
Each trial will compare how specific gene knockout salmon groups and wildtype salmon fry will respond to challenge with wildtype SAV3 isolate and/or attenuated SAV constructs that we have produced.
Harmfulness – The experiments will be severe – SAV3 will cause disease and mortality in fry.
Animals - The experiment will involve 10 times 680, i.e. 6800, Atlantic salmon fry (Salmo salar).
3R - The challenge trials will focus on the early infection kinectics, allowing them to be ended/terminated before SAV3-caused mortality will develop beyond the the sporadic. We will employ a salmon fry challenge model that we have developed in recent years, which we find more standardized than many other existing fish models. This constitutes a refinement of methodology, that will reduce the number of fish needed in each specific experiment.