In vivo transfer of a neuronal nitric oxide synthase expression vector into the rat bladder by electroporation.
OBJECTIVE
To investigate the possibility of in vivo gene transfer by attempting to transfer the neuronal nitric oxide synthase (nNOS) gene into rat bladder using electroporation.


METHODS
The bladder was exposed through an abdominal midline incision in 8-week-old male rats. Plasmid DNA of the marker genes green fluorescent protein (GFP) and luciferase, and the nNOS gene, was then injected into the subserosal space of the bladder and electroporation applied. At 72 h after gene transfer, GFP and luciferase were assayed in the isolated bladder and immunohistochemical staining used to detect nNOS; NO(x) released from isolated bladder strips was also assessed using microdialysis and high-performance liquid chromatography.


RESULTS
From the luciferase assay, 45 V, 1 Hz, 50 ms and eight pulses were selected as the optimum conditions for electroporation. Bladder specimens with GFP genes injected by electroporation showed bright and numerous sites of GFP expression in the smooth muscle layer. In rats with the nNOS gene injected by electroporation there was marked nNOS immunoreactivity, and NO(x) released from bladder strips was significantly greater than in the control groups.


CONCLUSIONS
These results suggest that electroporation is a useful technique for in vivo gene transfer into rat bladder smooth muscles, and that the nNOS gene transferred by this procedure functionally expresses and contributes to NO production.
Iwashita
H
H
Department of Urology, Graduate School of Medical Sciences, Kumamoto University, Japan.
Yoshida
M
M
Nishi
T
T
Otani
M
M
Ueda
S
S
D000818:Animals; D018076:DNA, Complementary / Q000378:metabolism; D018274:Electroporation / Q000379:methods*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008297:Male; D009119:Muscle Contraction; D009130:Muscle, Smooth; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D051381:Rats; D001743:Urinary Bladder / Q000201:enzymology*
D018076:DNA, Complementary; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C496322:Nos1 protein, rat