Techniques

We have a range of techniques to evaluate how the fish responds to invasion and damage. Some techniques, like qPCR, are very specific and targeted, providing us with detailed information on expression of certain genes. Other techniques, such as behavioural analyses, immunohistochemistry, histology and in vivo imaging give us insight into more general effects in the body.

Our overall aim is to obtain more knowledge on the immunological mechanisms and subsequent implications for the body. Using such approaches enable us to engineer novel and more cost-effective vaccines and obtain deeper understanding of immune responses. We have recently also implemented the CRISPR technique to be able to conduct functional studies on genes.

 

IHC is less used for zebrafish when compared to other fish such as rainbow trout and salmon. This is due to numerous available transgenic lines in zebrafish that can be used for functional studies. However, we work in both larvae and adult fish and especially in adults IHC can be very useful. We have antibodies against proliferative cells (PCNA), TCR, IgZ, Mpeg1, GFP, mCherry and a range of the pathogens we work with. It is possible to acquire more antibodies available for zebrafish on request.

Examples of investigations from our group using IHC in zebrafish can be found here:

Zebrafish (Danio rerio) as a model to visualize infection dynamics of Vibrio anguillarum following intraperitoneal injection and bath exposure.

Infection and immunity against Ichthyophthirius multifiliis in zebrafish (Danio rerio)

Antigen uptake during different life stages of zebrafish (Danio rerio) using a GFP-tagged Yersinia ruckeri.

 

 

 

 

 

 

 

 

 

 

Zebrafish, in their larvae and adult stages, where the tyrosinase gene has been knocked out with CRISPR/Cas9.

Zebrafish, in their larvae and adult stages, where the tyrosinase gene has been knocked out with CRISPR/Cas9. Photo by: Louise von Gersdorff Jørgensen and Moonika Haahr Marana.

We use the CRISPR/Cas technology for genome editing in zebrafish. We have created knock-out mutants for

genes encoding tyrosinase (tyr) and interferon regulatory factor 8 (irf8) to study certain biological function of the genes.

We inject embryos at the 1-cell stage to a create a mutation in the germ-line of zebrafish. The embryo injection generates the F0 line with mosaic mutation which we further outcross with the wild-type line to generate F1 heterozygous lines. Subsequently, we incross the F1 heterozygotes to obtain the F2 mutant line with homozygous mutation.

To check the efficacy of the genome editing we use heteroduplex mobility assay (HMA), T7 endonuclease 1 assay or sequence analysis of PCR amplicons.    

Learn more about our CRISPR work