Overexpression of nNOS by the Adenovirus Vector

Adenoviral vectors encoding rat nNOS (Ad.nNOS) or P-galactosidase (Ad.p-gal) were constructed by Channon et al. (20). They recombined an adenoviral vector system derived from the 340 mutant strain of adenovirus type 5, inserted a nuclear localizing P-galactosidase expression cassette in the E1 cloning region, and induced unique restriction sites at the 3' end of the E1 cloning site. The resulting viral vector (Ad.p-gal) served as a control virus. The Ad.nNOS vector was created by replacing the P-galactosidase cassette with one containing an nNOS cDNA. The rat nNOS cDNA was cloned into a pGEM CMV plasmid. This plasmid was modified and digested into a linearized fragment containing the left Ad5 inverted terminal repeat (ITR), cytomegalovirus (CMV) promotor, nNOS cDNA, and simian virus 40 (SV40) pA. The vector "backbone" DNA was a fragment with an ITR and the viral packaging signal at the extreme 5' end, which was prepared from the parent vector Ad.p-gal. These two fragments were ligated for 1 h at room temperature using T4 DNA ligase. The ligated DNA was transfected into 293 cells using the calcium phosphate method. Following the observation of a cytopathic effect, the recombinant Ad.nNOS was isolated and purified by double cesium chloride density cen-trifugation, as previously described (20,21). This recombinant Ad.nNOS vector contains a rat nNOS cDNA under the control of the CMV immediate-early promoter. The vector expresses functional nNOS protein in human vascular smooth muscle cells and human umbilical vein endothelial cells (20). The construct also expressed functional nNOS protein when infused in the carotid arteries of rabbits (21).

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