Cell lineage specific expression of artificial epitopes

The aim of the project is to develop a gene cassette system that can be used to be inserted into targeted alleles of murine embryonic stem (ES) cells generated by the European Conditional Mouse Mutagenesis (EUCOMM) or the Canadian gene targeting NORCOMM project. The gene cassette will consist of two genes oriented in tandem, a cre recombinase and a gene that will express an artificial epitope on the cell surface linked together via a 2A peptide. The 2A peptides are a preferred alternative to the internal ribosomal entry site as they cause the co-translational cleavage of proteins. The cassette will be inserted into targeted alleles of genes that are selectively expressed during the development of cell lineages in mice. Once inserted into the murine ES cells, mice can be generated, the lineage specific expression of the artificial epitope will allow the detection and selection of cells of a given cell lineage. Artificial epitopes are gene products not present in the genome of mice, expressed on the cell surface without signalling activity and selectable by specific monoclonal antibodies. Such cells can then be purified and further analysed. The insertion of the cre recombinase in the locus allows at the same time a genetic marking of cells derived from a given cell type for example by activating a loxP flanked gfp transgene. The combination of both systems allows to identify whether a given cell enters a cell lineage pathway and whether a given cell was part of a given cell lineage pathway although it might have lost the expression of the lineage specific marker. The project will demonstrate the feasibility of the approach by generating murine ES cells with T lymphocyte subset specific genes. In mice, generated from these ES cells we then will analyse Th1, Th2 and Th17 lineages in unchallenged mice and in mice challenged by infections.