Xui-Si Zhang1, Jing Huang, Cong-Qing Zhan2, Jing Chen, Tao Li3, Alan D Kaye, Sheng-Xi Wu, Lan Xiao3. 1. 1Department of Histology and Embryology, Chongqing Key Laboratory of Neurobiology, Third Military Medical University, Chongqing, China; Department of Neurobiology, The Fourth Military Medical University, Xi'an 710032, PR China. 2. Department of Neurobiology, The Fourth Military Medical University, Xi'an 710032, PR China. 3. Department of Histology and Embryology, Chongqing Key Laboratory of Neurobiology, Third Military Medical University, Chongqing, China.
Abstract
BACKGROUND: Many pain states are linked to central nervous system (CNS) diseases involving the dysfunction of dendritic arborization, making restoration a promising therapeutic strategy. Transfection of primary cortex neurons offers the possibility to study mechanisms which are important for the restoration of proper arborization. Its progress is, however, limited at present due to the lack of suitable gene transfer techniques. OBJECTIVE: To obtain better insight into the transfection potential of currently used techniques, 2 non-viral transfection methods, lipofection and gene electrotransfer (GET), were compared. STUDY DESIGN: This is a comparison study performed on cultured cells. METHODS: The transfection efficiency and neuronal viability, as well as the neuronal dendritic arborization after lipofection or GET, were compared. Primary cultured cortex neurons were transfected with the pEGFP-N1 plasmid, either using Lipofectamine 2000 (2, 3, or 4µL) or with electroporation, with our previously optimized protocol (200V/25 ms). RESULTS: Transfection efficiency and cell viability were inversely proportional for lipofection. The appropriate ratio of Lipofectamine and plasmid DNA provides optimal conditions for lipofection. Although GET offered higher transfection efficiency, it could not induce complex dendritic arborization, which made it unsuitable for in vitro gene transfer into cortex neurons. LIMITATIONS: Limitations include species variability and translational applicability for CNS diseases and pain states related to potential toxicity. CONCLUSIONS: Based on these findings, lipofection might be advantageous for in vitro application to primary cultured cortex neurons. Pain states, stress mediated pathogenesis, and certain CNS diseases might potentially utilize this important technique in the future as a therapeutic modality.
BACKGROUND: Many pain states are linked to central nervous system (CNS) diseases involving the dysfunction of dendritic arborization, making restoration a promising therapeutic strategy. Transfection of primary cortex neurons offers the possibility to study mechanisms which are important for the restoration of proper arborization. Its progress is, however, limited at present due to the lack of suitable gene transfer techniques. OBJECTIVE: To obtain better insight into the transfection potential of currently used techniques, 2 non-viral transfection methods, lipofection and gene electrotransfer (GET), were compared. STUDY DESIGN: This is a comparison study performed on cultured cells. METHODS: The transfection efficiency and neuronal viability, as well as the neuronal dendritic arborization after lipofection or GET, were compared. Primary cultured cortex neurons were transfected with the pEGFP-N1 plasmid, either using Lipofectamine 2000 (2, 3, or 4µL) or with electroporation, with our previously optimized protocol (200V/25 ms). RESULTS: Transfection efficiency and cell viability were inversely proportional for lipofection. The appropriate ratio of Lipofectamine and plasmid DNA provides optimal conditions for lipofection. Although GET offered higher transfection efficiency, it could not induce complex dendritic arborization, which made it unsuitable for in vitro gene transfer into cortex neurons. LIMITATIONS: Limitations include species variability and translational applicability for CNS diseases and pain states related to potential toxicity. CONCLUSIONS: Based on these findings, lipofection might be advantageous for in vitro application to primary cultured cortex neurons. Pain states, stress mediated pathogenesis, and certain CNS diseases might potentially utilize this important technique in the future as a therapeutic modality.