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.
Ning
Shou Bin
SB
Department of Gastroenterology, Air-force General Hospital, Beijing, China. Ningshoubin@hotmail.com
Zhang
Zhong Bing
ZB
Xie
Wei Fen
WF
Xin
Shuan Li
SL
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D002415:Cats; D002478:Cells, Cultured; D004947:Esophagus / Q000201:enzymology*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D009130:Muscle, Smooth / Q000201:enzymology; D019001:Nitric Oxide Synthase / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III; D010957:Plasmids; D014162:Transfection
C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III