Construction and characterization of murine neuroblastoma cell clones allowing inducible and high expression of the prion protein

A tetracycline-inducible expression system has been established for the pri on protein (PrP) in murine neuroblastoma cells (N2a), For this purpose, N2a cells were first stably transfected with either the tetracycline-controlle d transactivator or the reverse transactivator. After selection of N2a clon es which carried one of these transactivators, the murine PrP gene (Pmp) wa s introduced under the control of the transactivator-responsive promoter in a second round of stable transfection, Stably double-transfected N2a clone s carrying the reverse type but not the normal transactivator were found to be fully inducible, giving a low background of Pmp expression before induc tion and high expression after induction. Stably double-transfected N2a cel ls were at least as productive as N2a cells over-expressing Pmp permanently under the control of a strong viral promoter. Furthermore, the selected N2 a clones allowed the Pmp expression level to be quantitatively controlled b y varying the level of the effector substance, the tetracycline-derivative doxycycline, The clones were fully controllable, as over-expression could b e switched on and off as desired. These N2a clones may become an important tool for elucidation of the cellular function of PrP and may pave the way f or the tetracycline-inducible expression of many genes in this neuroblastom a cell line.