Nucleofection of cultured hippocampal neurons

为了了解大脑是如何运作的，我们必须能够记录在被称为神经元的特殊脑细胞中发生的电活动。为了对这些神经元进行电子记录，我们需要将它们从哺乳动物的大脑中移除。为了保持脑外神经元的存活，它们在孵化器中培养长达三周。我们要求的第一件设备是用于培养过程的层流引擎盖。如果没有这种设备，神经元就会感染环境中的细菌和病毒；一旦感染，它们就不能再用于实验。在成功培养后，我们需要改变目标蛋白的表达，以确定它们的功能。要做到这一点，我们需要将DNA和RNA导入神经元，这些DNA和RNA是我们有兴趣检测的蛋白质的密码。正确地对神经元进行转基因并维持细胞健康是非常困难的。然而，有一种相对较新的设备称为Amaxa Nucleofector，它可以在不损害细胞健康的情况下实现高效稳定的转基因。我们正在申请资金购买Amaxa核受体，以便我们能够研究抑制突触可塑性的细胞和分子机制。这项研究将使患有与正常衰老相关的记忆问题和神经疾病(包括癫痫和阿尔茨海默氏症)的加拿大人受益。

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'.