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KMID : 0613820070170020198
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Journal of Life Science
2007 Volume.17 No. 2 p.198 ~ p.203
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A Reliable Protocol for transfection of mature primary hippocampal neurons using a neuron-glia co-culture system
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Lee Hyun-Sook
Cho Sun-Jung
Jung Yong-Wook
Jin Ingn-Yol
Moon Ill-Soo
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Abstract
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DNA transfection is a powerful tool for studying gene functions. The Ca2+-phosphate precipitation remains one of the most popular and cost-effective transfection techniques. Mature neurons are more resistant to transfection than young ones and most other cell types, and easy to die if microenvironment changes. Here, we report a transfection protocol for mature neurons. The critical modifications are inclusion of glial cells in culture and careful control of Ca2+-phosphate precipitation under microscope. Cerebral glial cells were grown until ~70-80% confluence in DMEM/10% horse serum, which was thereafter replaced with serum-free Neurobasal/Ara-C, and E19 hippocampal neurons were plated onto the glial layer. Formation of fine DNA/Ca2+-phosphate precipitates was induced using Clontech CalPhos™ Mammalian Transfection Kit, and the size (0.5-1 ㎛ in diameter) and density (about 10 particles/100 ㎛²) were carefully controlled by the time of incubation in the medium. This modified protocol can be reliably applied for transfection of mature neurons that are maintained longer than two weeks in vitro, resulting in 10-15 healthy transfected neurons per a well of 24-well plates. The efficacy of the protocol was verified by punctate expression of pEGFP-CaMKIIα, a synaptic protein, and diffuse expression of pDsRed2. Our protocol provides a reliable method for transfection of mature neurons in vitro.
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KEYWORD
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Ca2+-phosphate precipitation, neuron-glia coculture, pEGFP-CaMKIIα, transfection
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