Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells
Optimized Transfection Strategy for Expression and Electrophysiological Recording of Recombinant Voltage-Gated Ion Channels in HEK-293T Cells
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DOI：
10.3791/2314
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发表时间：
2011-01-01
影响因子：
1.2
通讯作者：
Spafford, J. David
中科院分区：
文献类型：
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作者：
Senatore, Adriano;Boone, Adrienne N.;Spafford, J. David
在体外培养的人胚肾细胞（HEK-293 T）中重组电压门控离子通道的表达和电生理记录是一种普遍存在的研究策略。HEK-293 T细胞必须以足够低的密度接种到盖玻片上，使得它们彼此不接触，以便允许电生理记录，而不会由于与相邻细胞接触而产生混淆效应。转染的通道也必须在质膜上高效表达，用于全细胞膜片钳记录高于噪声水平的可检测电流。异源性离子通道在转染后通常需要在28 ° C下长时间孵育以实现足够的膜表达，但是在该温度下细胞-盖玻片粘附和膜稳定性的损失增加。为了解决这个问题，我们开发了一种优化的策略来转染和平板培养HEK-293 T细胞。该方法需要以相对高的汇合度转染细胞，并在转染后在28 ℃下孵育不同的孵育期，以允许足够的离子通道蛋白表达。然后将转染的细胞铺在盖玻片上，并在37 ℃下孵育数小时，这允许细胞刚性附着于盖玻片和膜再稳定。细胞可以在接种后不久记录，或者可以转移到28 ℃进行进一步孵育。我们发现，在转染后但在铺板前，在28 ℃下的初始孵育对于通常在质膜上表达不好的异源离子通道的有效表达是关键的。通过共表达的eGFP或从双顺反子载体表达的eGFP（例如，G. pIRES 2-EGFP），其含有正好位于内部核糖体进入位点上游的重组离子通道cDNA和eGFP编码序列。全细胞膜片钳记录需要专门的设备，加上抛光的记录电极和硼硅酸盐玻璃L形接地电极的制作。研究离子通道药理学的药物递送可以通过直接将药物微量移液到记录皿中来实现，或者通过使用微灌注或重力流系统来实现，所述微灌注或重力流系统在记录的细胞上产生不间断的药物溶液流。
The in vitro expression and electrophysiological recording of recombinant voltage-gated ion channels in cultured human embryonic kidney cells (HEK-293T) is a ubiquitous research strategy. HEK-293T cells must be plated onto glass coverslips at low enough density so that they are not in contact with each other in order to allow for electrophysiological recording without confounding effects due to contact with adjacent cells. Transfected channels must also express with high efficiency at the plasma membrane for whole-cell patch clamp recording of detectable currents above noise levels. Heterologous ion channels often require long incubation periods at 28 degrees C after transfection in order to achieve adequate membrane expression, but there are increasing losses of cell-coverslip adhesion and membrane stability at this temperature. To circumvent this problem, we developed an optimized strategy to transfect and plate HEK-293T cells. This method requires that cells be transfected at a relatively high confluency, and incubated at 28 degrees C for varying incubation periods post-transfection to allow for adequate ion channel protein expression. Transfected cells are then plated onto glass coverslips and incubated at 37 degrees C for several hours, which allows for rigid cell attachment to the coverslips and membrane restabilization. Cells can be recorded shortly after plating, or can be transferred to 28 degrees C for further incubation. We find that the initial incubation at 28 degrees C, after transfection but before plating, is key for the efficient expression of heterologous ion channels that normally do not express well at the plasma membrane. Positively transfected, cultured cells are identified by co-expressed eGFP or eGFP expressed from a bicistronic vector (e. g. pIRES2-EGFP) containing the recombinant ion channel cDNA just upstream of an internal ribosome entry site and an eGFP coding sequence. Whole-cell patch clamp recording requires specialized equipment, plus the crafting of polished recording electrodes and L-shaped ground electrodes from borosilicate glass. Drug delivery to study the pharmacology of ion channels can be achieved by directly micropipetting drugs into the recording dish, or by using microperfusion or gravity flow systems that produce uninterrupted streams of drug solution over recorded cells.