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A method of introducing large molecules into neurons.

一种将大分子引入神经元的方法。

基本信息

批准号：
09558103
负责人：
OHMORI Harunori
金额：
$ 8.13万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

It is desirable to introduce some marker molecules or biological active substances into neurons without any damage to the cell membrane. The technique should be easy to use and the target should be any individual neurons. We have manipulated the electroporation technique in order to apply it to a single neuron. We have utilized a small tip pipette filled with fluorescent dyes and pressed the tip against the cell membrane. With gentle application of negative pressure to the pipette a patch of cell membrane was swallowed into the pipette tip and then some voltage pulses of 0.1-1 Volt peak to peak of triangular waveform were applied within the pipette. These voltage pulses introduced neurons with fluorescence dyes. Molecules of 3k to 10k were successfully introduced and these neurons survived for next few days. However, we could not introduce much larger molecules into neurons such as plasmid DNAs. This is probably due to the fragility of neural membrane because we could introduce the same plasmid DNA into cos-7 by this technique. Since survival rate of neurons after microelectroporation was relatively high (maximum 50% overnight survival), we could still apply this technique in order to mark the neuronal pathway in the in vitro preparations. By marking neural tracts it should be easier to find and to record from the specific postsynaptic neurons. Although there are size limitation to this technique, it should also be useful to introduce some active substances into neurons to facilitate analyses of synaptic functions.

希望在不损伤细胞膜的情况下，将一些标记分子或生物活性物质引入神经元。该技术应该易于使用，目标应该是任何单个神经元。我们对电穿孔技术进行了操作，以便将其应用于单个神经元。我们使用了一个小的尖端移液管充满荧光染料，并压在细胞膜上。在移液管上轻轻施加负压，将一小片细胞膜吞入移液管尖端，然后在移液管内施加0.1-1伏的三角形波形峰到峰的电压脉冲。这些电压脉冲用荧光染料引入神经元。3k至10k的分子被成功引入，这些神经元在接下来的几天内存活。然而，我们不能将更大的分子引入神经元，如质粒dna。这可能是由于神经膜的脆弱性，因为我们可以通过这种技术将相同的质粒DNA引入cos-7。由于微电穿孔后神经元的存活率较高（过夜存活率最高可达50%），我们仍然可以应用该技术在体外制备中标记神经元通路。通过标记神经束，可以更容易地找到并记录特定的突触后神经元。虽然该技术存在尺寸限制，但在神经元中引入一些活性物质以促进突触功能分析也应该是有用的。