Stable expression of recombinant inducible nitric oxide synthase in NG108-15 cells and its biological characterization

The neuroblastoma x glioma NG108-15 cells were transfected with recombinant eukarytic expression plasmid pCMViNOS containing the full-length cDNA encoding inducible nitric oxide synthase (iNOS). A lot of G418-resistant clones were screened at 600 micrograms/ml of geneticin. In the 2# clone expressing iNOS gene, iNOS catalytic activity in the cytosol fraction displayed to have an increasing trend, accompanying with the accumulation of NO2- content in the supernantant of cultured cells and the intracellular cGMP concentration, which suggested that NO-cGMP signal pathway was mediated by the expression of iNOS gene and blocked by NG-nitro-L-arginine (L-NNA) and methylene blue (MB). Activity of iNOS was concentration-dependently inhibited by NOS inhibitors such as L-NNA and aminoguanidine. The result of measurement of NADPH diaphorase activity and immunocytochemical staining showed that localization of the function expression of iNOS protein mainly existed in the cytoplasm of NG108-15 cells transfected with pCMViNOS. Furthermore, the chromosomal integration, transcript and protein translation of foreign iNOS gene were identified by Southern hybridization, RT-PCR and Western blot, respectively. The results indicated that iNOS gene-transfected cells had mRNA transcription and specific protein expression at high level. Given the above results, the engineering cell line with stable expression of iNOS gene was successfully established. The new neuronal cell line may serve as a source of iNOS and provide a useful cell model for studying iNOS biological function and developing novel iNOS-selective inhibitors.