Susceptibility and immune responses of CRISPR-KO Atlantic salmon
Etterevaluering
The Norwegian Food Safety Authority must retrospectively assess all severe experiments.
Begrunnelse for etterevalueringen
This preliminary small-scale study successfully explored the roles of IgM and IFN-? in Atlantic salmon immunity using CRISPR knockouts F0 generation (CRISPants). Key objectives were met by: (1) generating preliminary survival data during acute (SAV3) and chronic (PRV1) viral infections, and (2) characterizing post-infection/vaccination immune responses in CRISPants.
Beyond primary goals, flow cytometry revealed reduced surface IgM expression in IgM-CRISPants, while gene expression analyses suggested compensatory mechanisms in IgM-deficient fish. These findings highlight the model’s potential for studying immune adaptation.
However, further validation using F1-generation CRISPants is needed to confirm observations, clarify mechanisms, and enhance biological relevance. This follow-up work will strengthen conclusions about salmon immune function and support practical applications (e.g., vaccine design and schemes, etc).
The minor variations in animal numbers were scientifically justified and did not increase experimental severity. Adjustments in Batch 1 pre-smolt numbers reflected technical refinements without added harm. Remaining Batch 2 pre-smolts (wildtype, albino, IgM-KO, and IFN-gamma KO F0 genetic groups) were preserved as broodstock to generate F1 offspring for future studies, prioritizing long-term research value over immediate use.
This study employed a 3Rs-compliant approach to investigate Atlantic salmon immune responses using CRISPR-generated models. While in vitro alternatives like CRISPR-edited cell lines exist, targeted in vivo studies remain essential for understanding complex whole-organism immunity involving multiple organs and cell types.
Sample sizes were carefully optimized to extract maximum data from the limited F0-generation CRISPants available, with future F1-generation studies planned to incorporate power analysis for more robust results. All experiments followed optimal vaccination and infection protocols that minimized animal distress and unnecessary pain and suffering. These included: (1) use of proven, standardized procedures, (2) rigorous (3x) welfare monitoring assessing both behavioral changes and established external clinical signs signs (hemorrhage, exophthalmia), and (3) implementation of clear humane endpoints that differentiated between temporary distress (e.g., body darkening) and irreversible decline (e.g., sustained loss of equilibrium). These protocols successfully balanced scientific requirements with animal welfare considerations throughout the study period.
Beyond primary goals, flow cytometry revealed reduced surface IgM expression in IgM-CRISPants, while gene expression analyses suggested compensatory mechanisms in IgM-deficient fish. These findings highlight the model’s potential for studying immune adaptation.
However, further validation using F1-generation CRISPants is needed to confirm observations, clarify mechanisms, and enhance biological relevance. This follow-up work will strengthen conclusions about salmon immune function and support practical applications (e.g., vaccine design and schemes, etc).
The minor variations in animal numbers were scientifically justified and did not increase experimental severity. Adjustments in Batch 1 pre-smolt numbers reflected technical refinements without added harm. Remaining Batch 2 pre-smolts (wildtype, albino, IgM-KO, and IFN-gamma KO F0 genetic groups) were preserved as broodstock to generate F1 offspring for future studies, prioritizing long-term research value over immediate use.
This study employed a 3Rs-compliant approach to investigate Atlantic salmon immune responses using CRISPR-generated models. While in vitro alternatives like CRISPR-edited cell lines exist, targeted in vivo studies remain essential for understanding complex whole-organism immunity involving multiple organs and cell types.
Sample sizes were carefully optimized to extract maximum data from the limited F0-generation CRISPants available, with future F1-generation studies planned to incorporate power analysis for more robust results. All experiments followed optimal vaccination and infection protocols that minimized animal distress and unnecessary pain and suffering. These included: (1) use of proven, standardized procedures, (2) rigorous (3x) welfare monitoring assessing both behavioral changes and established external clinical signs signs (hemorrhage, exophthalmia), and (3) implementation of clear humane endpoints that differentiated between temporary distress (e.g., body darkening) and irreversible decline (e.g., sustained loss of equilibrium). These protocols successfully balanced scientific requirements with animal welfare considerations throughout the study period.