Introduction of genes into living cells

One of our main subjects is an application of the FEL to gene therapy of genetic diseases, immunodeficiency syndromes and cancer. In this study, using the FEL, we tried to establish a model system for introducing genes into the stem cells from which all blood cells are derived. Our aim is to specifically mark the stem cells with monoclonal antibodies which are conjugated with efficient FEL absorbers. Cells are then irradiated with FEL at a wavelength corresponding to the absorption energy of the absorber. We speculate that the gap formation of cell membrane will occur, caused by the thermal shock due to the absorption of the FEL energy. In the preliminary experiments, Green Fluorescent Protein (GFP) genes were used as an indicator gene for evaluating irradiation conditions and gene expression in Cos-1 cells (kidney, African green monkey). By irradiation of 6.1 μm FEL with the power density of 50 mW/mm2 (av.), we could successfully introduced the GFP genes into Cos-1 cells as detected by a fluorescent microscope. As an animal model for gene therapy, we tried to transfer the RAG-2 genes into hematopoietic stem cells from RAG-2 deficient mice, which have severe immunodeficiency because the lack of RAG-2 gene required for lymphocyte development. As the results by this construct, the infant lymphocytes (T cells and B cells) could be observed in the thymus of the RAG-2 deficient mice 2 weeks post operative. (author)