T cells are pivotal for the generation and regulation of antigen-specific immune responses, but can also cause autoimmune diseases, such as multiple sclerosis. A milestone for multiple sclerosis research was the discovery that effector memory CD4+ T cellsCD4 T cells reactive against central nervous system (CNS) antigens were the cellular cause of the disease in the animal model experimental autoimmune encephalomyelitis (EAE). Since then, protocols have been developed to generate stable, oligoclonal effector T cells reactive against CNS antigens and therefore capable of inducing EAE from primary cultures. Importantly, the discovery that antigen-specific T cells can be efficiently transduced by retroviral vectors without interfering with T cell function made it possible to engineer these cells with a variety of genes, such as fluorescent protein and sensors, that have proven to be powerful tools to uncover T cell function in vivo and in vitro. In this chapter, we provide protocols for the generation of primary effector memory T cell lines from the Lewis rat against CNS and control antigens and for their transduction with genes of interest.