Nucleofection of cultured hippocampal neurons

培养的海马神经元的核转染

• 批准号: 390105-2010
• 负责人: Woodin, Melanie
• 金额: $ 1.79万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments - Category 1 (<$150,000)
• 财政年份: 2009
• 资助国家: 加拿大
• 起止时间: 2009-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=443110
• 关键词: Nucleofection; cultured; hippocampal; neurons

In order to understand how the brain functions we must be able to record the electrical activity occurring in specialized brain cells called neurons. In order to make electrical recordings from these neurons we need to remove them from the mammalian brain. To keep the neurons alive outside of the brain, they are cultured in an incubator for up to three weeks. The first piece of equipment we are requesting is a laminar flow hood, which is used in the culturing process. Without this piece of equipment the neurons become infected with bacteria and viruses found in the environment; once they are infected they can no longer be used for experiments. Following successful culturing, we need to alter the expression of proteins of interest in order to determine their function. To do this we need to transfect neurons with DNA and RNA that code for the protein we are interested in examining. It is difficult to transfect neurons properly and maintain cell health. However, there is a relatively new piece of equipment called the Amaxa Nucleofector, which achieves high-efficiency stable transfection with no compromise in cell health. We are requesting funds to purchase an Amaxa Nucleofector to allow our investigation of the cellular and molecular mechanisms underlying inhibitory synaptic plasticity. This research will benefit Canadians who suffer from memory problems associated with normal aging and neurological disorders, including epilepsy and Alzheimers'.

为了了解大脑的功能，我们必须能够记录在称为神经元的专门脑细胞中发生的电活动。 为了从这些神经元中进行电子记录，我们需要将它们从哺乳动物的大脑中取出。 为了保持神经元在大脑外存活，它们在培养箱中培养长达三周。 我们要求的第一件设备是层流罩，用于培养过程。 如果没有这种设备，神经元就会被环境中的细菌和病毒感染;一旦被感染，它们就不能再用于实验。 成功培养后，我们需要改变感兴趣的蛋白质的表达，以确定它们的功能。 要做到这一点，我们需要用编码我们感兴趣的蛋白质的DNA和RNA来检测神经元。 很难正确地切除神经元并保持细胞健康。 然而，有一种相对较新的设备称为Amaxa Nucleofector，它可以实现高效稳定的转染，而不会损害细胞健康。 我们正在申请资金购买Amaxa Nucleofector，以便研究抑制性突触可塑性的细胞和分子机制。这项研究将使加拿大人受益，他们患有与正常衰老和神经系统疾病有关的记忆问题，包括癫痫和老年痴呆症。