The scissors had successfully boarded the cells and worked its magic there: It had precisely located the gene selected by Zoppo on the rainbow trout genome, changed the DNA sequence there and created a so-called knock-out mutation in the gene. This means that due to the mutation in the DNA sequence, the gene could now no longer carry out its usual role in the cell: an excellent experimental system for the researcher to study this very role of the gene. "We observe what happens when a cog in the system fails. In this way, so far poorly understood gene functions can be elucidated," explains Zoppo.
However, there was a small problem. It is one of the properties of the CRISPR/Cas9 system that a gene can be mutated as desired, but: The exact DNA sequence that is created in the process is randomly formed in each cell. "Because we had used several cells for the experiment, each cell now had its own DNA sequence," says Zoppo. The researcher's task was now to pick out a "mother cell" from these and multiply it so that a cell line of genetically identical daughter clones was created. "Here, however, the fish cells again proved to be astonishingly unconventional," says Zoppo. "None of the conventional and widely used methods I knew and tried were applicable here. It just didn't work," says Zoppo.
The breakthrough finally came with an older method using simple cloning cylinders. When cells multiply in culture, they form colonies around the mother progenitor cell, which can be isolated with the cylinder and transferred to another plate. "It's actually quite simple once you get the hang of it," says Zoppo.