Figure 1.

Rapamycin-inducible production and secretion of GDNF. (a) Lentiviral constructs: The transactivator expression vector (TEV) mediates constitutive expression of the fusion proteins NLS-FRB-p65 and NLS-ZFHD1-3xFKBP allowing inducible transcription from an engineered minimal interleukin 2 promoter (P_{IL-2 min}). P_{IL-2 min} is used in the glia derived neurotrophic factor (GDNF) expression vector to produce the fusion protein SS-4xFKBP-36M-FCS-GDNF providing rapamycin-regulated secretion of GDNF.⁹ LTR, ψ, and Flap sequences are derived from HIV-1 (the long terminal repeats, the packaging sequence, and the central Flap element, respectively). P_CMV is the CMV promoter, WPRE the Woodchuck hepatitis virus responsive element, and ECMV IRES an internal ribosome entry sequence from the encephalomyocarditis virus. Under the conditions used for cell amplification (i.e., in the absence of inducer) only the fusion proteins NLS-FRB-p65 and NLS-ZFHD1-3xFKBP were produced from TEV. (b) Cell clones derived from HEK 293T, D407, and HER 911 cells displaying inducible production of GDNF. Cells (5 × 10⁴) were cultivated in the presence and absence of 10 nmol/l of the rapamycin derivative AP 21967. Values (±SE, n = 3) indicate the amounts of GDNF in the medium after 2 days of culture.