Gene transfer to intact mesenteric arteries by electroporation.
The purpose of the present study was to develop a rapid, reproducible method of nonviral gene transfer to the intact vasculature. Male Sprague-Dawley rats were anesthetized, a midline abdominal incision was made and segmental branches of the superior mesenteric artery were dissected free of surrounding mesentery. A specially designed electroporation probe was placed around the neurovascular bundle and the electroporation chamber filled with a solution containing the firefly luciferase expressing plasmid (pCMV-Lux-DTS) or the green fluorescent protein expressing plasmid (pEGFP-N1). Vessels were electroporated with eight 10-ms pulses of 200 V/cm. Sixty seconds after electroporation, the DNA solution was removed, the intestine returned to the abdomen and the abdominal wall closed with suture and metal wound clips. Six hours to 5 days later, rats were sacrificed and electroporated vessels were recovered. Luciferase activity of the blood vessels was monitored. Gene expression was detected as early as 6 h postelectroporation, peaked at 1-3 days with levels up to 1 ng of reporter gene product per vessel segment and returned towards baseline by day 5. Histological analysis of blood vessel segments revealed green fluorescent protein-positive cells throughout the thickness of the vessel wall (endothelial cells to adventitia). Responses of electroporated vessels to vasoconstricting stimuli were indistinguishable from those of control vessels at either 2 or 40 days posttreatment. The results of this study provide evidence that electroporation is an effective means for introducing naked DNA into the blood vessel wall and form the basis for future studies on targeted gene therapy to the intact vasculature.
Martin
J B
JB
Department of Physiology, University of South Alabama College of Medicine, Mobile, AL 36688, USA.
Young
J L
JL
Benoit
J N
JN
Dean
D A
DA
D000818:Animals; D018274:Electroporation; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D007700:Kinetics; D008156:Luciferases / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D008638:Mesenteric Arteries / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D014018:Tissue Distribution; D014162:Transfection / Q000379:methods*; D014661:Vasoconstriction
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D008156:Luciferases