Stable transfection of cloned murine T helper cells

Abstract

Here we describe a protocol for the stable transfection of murine T helper (Th) cells and long term culture of the resulting transfectants. The electroporation protocol was established for the murine Th2 clone L1/1 by testing different parameters determining the electric field (capacitance, voltage, single or twin pulse) as well as the activation status of the cells. The transfected T cells were genetically altered by stable integration of the neomycin resistance gene, encoded in the vector pM5neo, into the genome. For selection and long term culture of stable transfectants a scheme combining selection with the antibiotic neomycin (G-418, Geneticin) and repeated stimulation with antigen presenting cells (APC) and antigen was established. This protocol should also be applicable to other antigen reactive T cells. The resistance of the T cells to neomycin correlated directly with expression of the transferred neomycin resistance gene as demonstrated by mRNA analysis. Applying periodic reselection with neomycin the transfected Th2 cells were found to be stable for more than 18 months in culture and displayed an unaltered antigen recognition and lymphokine production pattern as compared with the untransfected L1/1 Th2 cells.