Assessing transgene incorporation into cultured Atlantic salmon cell genomes.

Abstract

Aquaculture’s significance commences and rapid growth in the 1970s has made it the fastest-growing food production technology. However, the production of an economical, environmentally, and socially sustainable aquaculture comes with some major challenges. One of these is infectious diseases in both the industry and in the wild. Not only does this have a negative impact on the animal’s health and welfare, but its mortality also represents a significant economic loss for the producers. Norway has a contribution of over 50% to the Atlantic salmon production and is therefore affected by these challenges. Potential ways to decrease the mortality rates and better the Atlantic salmon’s welfare is to understand how the genes associated with disease resistance work and adapt this knowledge into gene editing. A potential source for research of these possibilities is the use of cell lines derived from Atlantic salmon. This opens a source for in vitro research that can provide tools and strategies used to control diseases. However, due to Atlantic salmon’s genome duplication, it’s more challenging to successfully genome edit them. With new technologies such as reprogrammable CRISPR/Cas9, this challenge could potentially be resolved. In order to achieve a continuous Cas9-expressing cell line, the optimal MOI needs to be found for each cell line. One way to find this is by accurately and precisely quantifying the copy number of transduced cells to modify the titer. This thesis shows the development of qPCR assays as a method to estimate the copy number of integrated DNA target molecules in two Atlantic salmon cell lines; ASG-10 and ASSF. Detection of amplified templates was accomplished with SYBR Green 1 and carefully validated primer pairs. Multiple evaluation steps were conducted prior to the final validation of a primer pair for each target sequence. In the initial evaluation, the primers were tested on serially diluted targets in a qPCR assay that was used to generate standard curves, which assessed their efficiency and performance. Afterward, their specificity was evaluated through a melt curve analysis and further supported by gel electrophoresis. Only primer pairs with efficient, well-performing, and specific results were used as external standards in the quantification assay. Standard curves generated from the validated primers were used to quantify the copy number of target DNA molecules in unknown samples.