INTRODUCTION: Our previous studies have established that EcR-293 cells can be genetically engineered to release NGF in vitro up to 9 days following induction with Ponasterone A (PonA). To further tightly regulate the NGF release, we incorporated herpes simplex virus-thymidine kinase (HSV-TK) gene as a molecular “off switch” in these cells. The present study was performed to assess the reduction in NGF expression following exposure to the suicide gene.
MATERIALS AND METHODS: HSV-TK gene was cloned in expression cassette of plasmid vector pcDNA (pcDNA-TK). NGF producing human embryonic kidney cells (hNGF-EcR-293) cells were transfected with pcDNA-TK to produce a stable cell line named as hNGF-EcR-293-TK. These cells induced with PonA(+) and PonA(-) were left for 5 days without changing the medium. On day 5 gancyclovir was added to the experimental wells. Supernatants were collected at 1, 2, 3 and 4 days post gancyclovir treatment and tested for NGF expression and bioactivity by ELISA and PC-12 cell assay, respectively.
RESULTS: A stable cell line, hNGF-EcR-293-TK was established by transfecting HSV-TK in NGF producing EcR-293 cells followed by cloning and expanding a single cell clone which was used for molecular switch off experiments. Till day 5 post induction with PonA, there was an increase in NGF production. The treatment with gancyclovir at day 5 post induction resulted in the progressive decline in NGF production, reduced PC-12 bioactivity and cell killing. Non-transfected hNGF-EcR-293 and non-induced hNGF-EcR-293-TK control cells did neither had NGF expression nor exhibited any PC-12 bioactivity and did not exhibit any cell killing to gancyclovir treatment. Further experiments are in progress.
CONCLUSIONS: Our current study successfully established a cell line which is capable of both producing and switching off NGF production whenever needed enabling us to have a tighter and regulated control of NGF expression.