Genetically modified cells hold great promise in several fields of biomedicine, starting from anti-tumour therapy and going as far as the treatment of autoimmune diseases, the rejection of transplanted organs or the treatment of fibrosis. The genetic modification of the cells includes the expression of a protein, such as, for example, chimeric antigen receptors in T cells or in macrophages. It can also be used for inactivating the protein expression, most easily by RNA interference or by CRISPR/Cas9 technology.
Retroviral and lentiviral vectors are crucial tools for genome modification in research. They are essential especially for genetic manipulation of primary cells and cell-lines which are very difficult to transfect. They are routinely used for:
- Expression of recombinant proteins, with or without tags (e.g. 6His-, FLAG-, c-myc-tags for protein purification, GFP-, mCherry-, CFP-, YFP- tag for cell imaging).
- Protein depletion by RNA interference (lentiviral particles coding for shRNA)
- Gene knock-out by CrispR/Cas9 technology.
Our facility proposes:
- Advice for the choice of the viral vector to use according to the targeted cell type (e.g. lentiviruses transduce both non-dividing and actively dividing cell types, while standard retroviruses can only infect mitotically active cell types).
- Strategy for molecular cloning of desired cDNA in the viral plasmids (several plasmids available, with fluorescent or antibiotic selection markers).
- Molecular cloning and purification of high-quality plasmid DNA for virus production.
- Viral production and subsequent transduction of the target cell.
- Gene inactivation via CRISPR/Cas9 ribonucleo-protein complexes in primary cells.
Workflow of virus production (created with bio-render).
Image of CAR-T cells (Green) produced in our facility, eating a tumor cell (Blue).