Delivery of DNA into bladder via electroporation.
The possibility of in vivo gene transfer into the rat bladder by electroporation (EP) was evaluated. The bladder was exposed through an abdominal midline incision in 8-week-old male rats. Plasmid DNA of marker genes, green fluorescent protein (GFP) and luciferase, and the neuronal nitric oxide synthase (nNOS) gene were then injected into the subserosal space of the bladder and EP was applied. At 72 h after gene transfer, GFP and luciferase were assayed in the isolated bladder, and immunohistochemical staining was used to detect nNOS. NOx released from isolated bladder strips was also assessed using microdialysis procedure. From the luciferase assay, 45 V, 1 Hz, 50 ms, and 8 pulses were selected as the optimum conditions for EP. Bladder specimens with GFP genes injected by EP showed numerous bright sites of GFP expression in the smooth-muscle layer. In rats with the nNOS gene injected by EP, there was marked nNOS immunoreactivity, and NOx released from bladder strips was significantly greater than that in the control groups. These results suggest that EP 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.
Yoshida
Masaki
M
Department of Urology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan.
Iwashita
Hitoshi
H
Otani
Masayuki
M
Masunaga
Koichi
K
Inadome
Akito
A
D000818:Animals; D004274:DNA, Recombinant / Q000008:administration & dosage* / Q000235:genetics; D053672:Electrochemotherapy / Q000379:methods*; D018274:Electroporation / Q000379:methods; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000008:administration & dosage / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D001743:Urinary Bladder / Q000378:metabolism*
D004274:DNA, Recombinant; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D009569:Nitric Oxide; D008156:Luciferases; D052248:Nitric Oxide Synthase Type I; C496321:Nos1 protein, mouse