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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)
 
 
 

Construction of a recombinant adenovirus carrying endothelial nitric oxide synthase gene and its expression and control in esophageal smooth muscle cells.

OBJECTIVE: Nitric oxide (NO) is a major inhibitory neurotransmitter, and its deficiency plays an important role in the pathogenesis of motility disorders of the gastrointestinal tract. The present study was designed to generate a recombinant adenovirus containing the tetracycline (Tet)-regulated endothelial nitric oxide synthase (eNOS) gene and to detect the controllable expression of the gene in esophageal smooth muscle cells (ESMC). METHODS: The construction of the recombinant adenovirus was completed in three steps: (1) a Tet-responsible expression cassette was made by cloning the full-length cDNA encoding eNOS into a pTRE-Shuttle Vector, which can be regulated by tetracycline or its analogs, such as doxycycline (Dox); (2) the expression cassette was transferred to Adeno-X viral DNA to form a recombinant adenoviral plasmid (pAd-eNOS) by means of an in vitro ligation reaction; and (3) the Ad-eNOS was packaged into infectious adenoviral particles (Adeno-X-TRE-eNOS) by transfecting human embryonic kidney (HEK) 293 cells. Cultured ESMC were coinfected by Adeno-X-TRE-eNOS and regulation virus (Adeno-X Tet-off virus), and the Dox-regulated eNOS expression was detected by RT-PCR and western blot. RESULTS: The recombinant adenovirus (Adeno-X-TRE-eNOS) was generated successfully by an in vitro ligation reaction. The expression of the eNOS gene in the coinfected ESMC was confirmed by RT-PCR and western blot. Furthermore, the transcription could be precisely regulated in a dose-dependent manner in a series of concentrations of Dox, and it was completely turned off when the concentration reached 0.01 microg/mL. CONCLUSIONS: A Tet- (or Dox-) regulated recombinant adenovirus carrying eNOS was successfully generated and controllable expression of eNOS in ESMC was achieved, which provides some material for conducting further gene therapy studies with eNOS.[1]

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