LeGO-Vectors & RGB-Marking: Lentiviral tools for cell manipulation and multi-colour clonal tracking of stem and tumour cells Event

LeGO Vectors

Today lentiviral vectors are a very common tool in many laboratories seeking stable modification of cells. A few years ago we combined this type of vector with all available colours of fluorescent proteins, many different drug resistances and luciferases, ready to transfer shRNAs or cDNAs into your target cells. It is even possible to combine all those features on a single, modular construct, following the principle of building blocks. This set of vectors was named LeGO vectors.

RGB Marking

Based on the already quite colourful set of LeGO vectors we established a new method of clonal cell labelling, able to generate virtually all perceivable colours: RGB Marking. After transplantation of e.g. stem or tumour cells, it is a convenient method to track these cells by means of stably expressed fluorescent proteins – in case of RGB Marking with a resolution down to even single cells with unique colour labels. In comparison to whole tissues labelled with one and the same label (e.g. GFP only), RGB Marking represents a strategy to follow-up the fate of individual cells and their progeny.

RGB Marking developed in our lab allows to stably stain individual cells with a characteristic colour hue. Using this method we have shown that clonal colours remain stable over time, in all daughter cells, even upon (re)transplantation. The general principle of RGB colour mixing is broadly known from computer or TV screens, however in RGB Marking the colours originate from mixing three fluorescent proteins in the basic colours red, green and blue (RGB) at different but very constant amounts in individual cells, simultaneously transduced by just three lentiviral LeGO vectors. Individually marked cells or clones can be tracked to assess the behaviour of cells in different situations, e.g. loss of clonality during tumour progression, clonality of metastases or numbers of engrafted stem cells within a transplant.

We have established novel vectors and methods to further improve applicability of RGB Marking. LeGO vectors with drug-selectable fluorescent markers allow for convenient protocols to obtain pure, RGB-marked cell populations in a short time frame. We also explored different vector types, utilized various promoters and incorporated novel fluorescent proteins to identify optimal RGB-marking strategies for distinct experimental settings. Finally we investigated the possibility to apply the RGB vectors in vivo to stain cells in situ. This approach allows to follow newly generated cells and track their migration without needing transgenic animals or ex-vivo handling of (stem) cells.