Tissue-engineered constructs offer a new hope to patients suffering from functional impairment after nerve injury. An effort has been made to focus on delivery, regulation, and "molecular shutoff" of nerve growth factor (NGF) in tissue-engineered constructs. We have previously demonstrated that human embryonic kidney (HEK-293) cells can be genetically modified to secrete NGF at varying time points upon up regulation with Ponasterone A (PonA) both in vitro and in vivo. In the present study, HEK-293 cells that stably and inducibly produce NGF were further stably transfected with herpes simplex virus-thymidine kinase gene as a suicide gene (hNGF-EcR-293-TK) in order to shut off the NGF secretion and kill the cells upon treatment with ganciclovir (GCV). These cells following induction with PonA secreted NGF levels of 6659.2 +/- 489.4 pg/mL at day 10 postbooster dose at day 5, which was significantly higher than the control noninduced cells. The NGF secreted by these cells was bioactive as determined by a rat adrenal pheochromocytoma (PC-12) cell bioassay. Treatment of these cells with GCV significantly reduced the NGF levels to 645.3 +/- 16.2 pg/mL at day 10 and live cell numbers dropped to 7.95 x 10(3) +/- 278 compared to 2.73 x 10(5) +/- 6.1 x 10(4). GCV-treated cell media when transferred to the PC-12 cell bioassay demonstrated less than 10% cells differentiating into neurite-like extensions. We conclude that hNGF-EcR-293-TK cells can inducibly secrete bioactive NGF when treated with the inducing agent and can also be killed upon treatment with GCV. This double-gene transfection for gene expression and molecular shutoff mechanism will be a useful tool in tissue-engineered nerve constructs.