Methods in virology and molecular biology
At the department of clinical microbiology who hosts our research group, we have access to all the infrastructure needed to culture cells, produce viruses, and carry out molecular biology experiments. These are needed not only to support the production of materials for our biophysical experiments, but also to verify that observations made on our biomimetic platforms can be linked to observations made in living cells. We also have access to both BSL-2 and BSL-3 areas, currently our key instruments are all in BSL-2 areas meaning they can be used to study infectious materials.
Within our research activities, we regularly produce and purify viruses for our experiments. Purified viruses can be titrated by plaque assay or quantified by qPCR, immunofluorescence-based microscopy or flow cytometry. In addition, our group is equipped with lentivirus and VSV-based systems to generate pseudotype virus particles, which enables us to study the surface proteins of some severe viruses such as Ebola virus or SARS-Cov-2 more directly and safely. Generated pseudotype virus particles can be quantified by qPCR or flow cytometers. With the help of cloning techniques, we can also perform plasmid construction, such as introducing designed mutations into plasmids to tailor them to our experimental needs. Modified materials are then characterized through PCR, western blots, and/or co-immunoprecipitation.
As with our library of cell membrane mimics, we are also continuously expanding the library of cell types in the group. With the help from our neighbor groups, it is possible to generate special cell lines with certain genes knocked in/out or knocked down via the application of siRNA or Caspase 9 techniques. Additionally, we plan to use more complex in vitro systems, for example to produce a complete and thick glycocalyx. Through collaborations we also work with materials from human enteroids. Finally, our location at the hospital allows us easier access, through collaborations with our colleagues, to patient samples for use in our research.