Electroporation for direct spinal gene transfer in rats.
We investigated the feasibility of delivering exogenous genes into spinal cord using direct in vivo electrotransfection. Gene transfer to the spinal cord was accomplished via direct intrathecal injection of pE-GFP C1 vector, followed by five electric pulses for 50 ms at 200 V delivered intrathecally. The spinal cords were retrieved and analyzed with fluorescence microscopy, reverse transcription polymerase chain reaction (RT-PCR), and Western blotting. At day 1, 3 or 7 following electroporation a clear GFP expression in spinal cord tissue was detected. The most prominent transfection occurred in the meningeal cells and superficial layer of the spinal cord. Successful transfection was also confirmed with RT-PCR and Western blotting. The expression of GFP protein was peaked between 3 and 7 days after electroporation and significantly decreased at 14 days. No behavioral or spinal neurodegenerative changes were detected at any time point. This study demonstrates that direct in vivo electrotransfection represents an effective and simple method for spinal gene delivery and have a potential to be used clinically, especially, acute or chronic pain.
Lin
Chung Ren
CR
Department of Anesthesiology, Chang Gung Memorial Hospital, 123 Ta-Pei Road, Niao Shung Hsiang, Kaohsiung Hsien, 833 Taiwan.
Tai
Ming Hong
MH
Cheng
Jiin Tsuey
JT
Chou
An Kuo
AK
Wang
J J
JJ
Tan
Ping Heng
PH
Marsala
Martin
M
Yang
Lin Cheng
LC
D000818:Animals; D018274:Electroporation / Q000295:instrumentation / Q000379:methods*; D005455:Fluorescent Antibody Technique; D015316:Genetic Therapy / Q000295:instrumentation / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000627:therapeutic use*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000378:metabolism; D007278:Injections, Spinal; D008159:Lumbar Vertebrae; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D008578:Meninges / Q000166:cytology / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D017288:Pain Threshold / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley / Q000235:genetics / Q000601:surgery*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013116:Spinal Cord / Q000166:cytology / Q000378:metabolism / Q000601:surgery*; D013126:Spinal Nerve Roots / Q000166:cytology / Q000378:metabolism; D014162:Transfection / Q000295:instrumentation / Q000379:methods*
D005904:Glial Fibrillary Acidic Protein; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins