Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons
Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons
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DOI：
10.3791/2373
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发表时间：
2011-01-01
影响因子：
1.2
通讯作者：
Dent, Erik W.
中科院分区：
文献类型：
--
作者：
Viesselmann, Christopher;Ballweg, Jason;Dent, Erik W.
海马和皮质神经元已被广泛用于研究中枢神经系统（CNS）神经元极化、轴突/树突生长以及突触形成和功能。培养这些神经元的一个优点是它们容易在非常低密度的二维基质上生长，形成独特的轴突和树突。这种特性使它们在确定神经元发育的许多方面非常有用。此外，通过为这些神经元提供胶质调节，它们将继续发育，形成功能性突触连接并在培养物中存活数月。在本方案中，我们概述了一种技术，解剖，培养和切除胚胎小鼠海马和皮层神经元。转染通过在经由核转染铺板之前将DNA电穿孔到神经元中来完成。该方案具有在发育早期（类似于接种后4- 8小时）表达荧光标记的融合蛋白的优点，以研究蛋白在极化、轴突生长和分支期间的动力学和功能。我们还发现，在铺板之前的这种单次转染将荧光标记的融合蛋白表达维持在适合于在神经元的整个寿命（培养中> 2个月）中成像的水平。因此，这种方法是有用的研究蛋白质定位和功能在整个中枢神经系统的发展，很少或没有破坏神经元功能。
Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (similar to 4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (> 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.