The expression of rat brain constitutive nitric oxide synthase in NG108-15 cell

Full length cDNA of rat brain cNOS was inserted into the polylinker area of pRC/CMV with specific orientation and an eukaryocyte expression vector pCMVcNOS was obtained. The existence of cNOS gene was demonstrated by PCR amplification, using pCMVcNOS gene as the model and primers designed in accord with the internal sequence of cNOS gene. The insertion and orientation of pCMVcNOS were further verified by enzymatic cleavage. NG108-15 cells were transfected with pCMVcNOS by calcium phosphate DNA coprecipitation and lipofectin transfection. G418 resistant monoclonal cells were selected with a culture medium containing 600 micrograms/ml G418. NOS activity of each clone was assayed by monitoring the conversion of 3H-Arginine to 3H-Citrulline. High expression cell lines were selected through measurement of the cytosol and particulate NOS activity. Out of 42 resistant monoclonal cell lines, 3 stable high expression clones have been finally selected. The increase of expressed cytosol NOS was more obvious. The result showed that the cell lines expressing cNOS at a high level had been obtained.