Name: controllable ion channel expression through inducible transient transfection
Uploaded: 2017-02-17T15:00:00
Description: Watch this Scientific Journal Video about Controllable Ion Channel Expression through Inducible Transient Transfection at JoVE.com

这项工作是由以色列科学基金会[资助1721至1712年，1368年至1312年，以及1444年至1416年（到AP）的支持。 AP隶属于Brettler中心和David R.布鲁姆中心，药学院，耶路撒冷希伯来大学。

1.结扎兴趣的基因到载体阻遏网站<ol><li>获得诱导载体如的pcDNA5 / FRT / TO或pcDNA4 / TO。 </li><li>通过在硅片 DNA分析软件4分析在该还设有在所选择的载体的多克隆位点的基因的中间任何潜在易感限制性位点感兴趣的基因。 </li><li>使用标准的重叠PCR技术4中 ，侧翼具有两个选择限制性内切酶识别位点（即未在感兴趣的基因中发现）感兴趣的基因，第一起始密?...

<ol>
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转染蛋白表达和研究一种广泛使用的协议，与许多不同的变化，以提高表达的一致性和稳定性。瞬时转染试剂提供一种简单，易于使用的协议，其中感兴趣的细胞和蛋白质可在数小时内从转染时进行分析以过夜。不幸的是，当分析模式需要蛋白的表达，如电2单声道录音的水平一致这种做法是不可预知的。可替代地，四环素阻遏表达系统下的稳定细胞株已开发提供了一种方法，以...

异源表达系统是最广泛使用的技术来研究细胞功能1众多之一。其低的内源性蛋白图谱，最少的维护要求，可靠的增长，并采取和表达外源DNA的能力已使细胞系，如人类胚胎肾（HEK293）和中国仓鼠卵巢（CHO）几乎是必不可少的，以生物学研究2，3。采用异种系统的研究领域包括膜蛋白，细胞内信号和酶活性。以下外源DNA的转染进入细胞内，许多不同形式的分析可以被执行，包括电，比例钙成像，免疫印迹等 4，5。 由于广泛的潜在应用为异源表达系统中，许多各色核苷酸的试剂和产品已经发展到利用这些细胞和它们的质量6。外国DNA暂时或永久地融入细胞研究外源蛋白的DNA递送系统已成为生物学研究最流行和最有用的工具之一。更具体而言，瞬时转染的DNA导入细胞被广泛地用作一种简单，直接的过程，需要相对少的时间和材料。另外，该经过转染的细胞的成功率很高7。这种技术是非常可靠的，当用一个标记基因诸如绿色荧光蛋白（GFP）组合，并且可用于许多不同的技术，如钙成像和电5。不幸的是，虽然，瞬时表达的DNA导入宿主细胞带有一些重大的缺陷，而不是在每个细胞中的表达水平是不可靠的最少。质粒DNA的拷贝数采取ū每个细胞p是不可控的，因此个别实验之间的表达式差别很大2。当任一试图复制生理条件下，或在执行精确的数据收集技术这个问题变得显著。 作为解决上面提到的，稳定转染的协议已经被设计的复杂，其中感兴趣的基因可以被插入到可诱导启动子，的严密控制下的细胞的基因组，如四环素阻遏表达系统，确保单该质粒的拷贝整合到每个细胞的基因组和转录机制诱导后仅表示，例如，在多西环素的存在。虽然这解决了不一致蛋白表达水平的障碍，该方法失去瞬时转染的快速和相对简单的协议的便利性。建立一个稳定的细胞系发生在whic至少几周高电平单必须校准杀灭曲线由特定抗生素设定为保持蛋白质表达并确保载体整合并巧妙地选择和生长的细胞集落。总的来说，这需要显著更多的时间和精力用较低的成功率8。 在这里，我们引入两个流行的转染方案的长处绘制，以提供一个简单而有效的方法来控制在任何可诱导的细胞系的表达水平的中间的协议。同时保持与可诱导的tet系统细胞，我们瞬时转染我们感兴趣的基因，瞬时受体电位阳离子通道亚家族V部件1（TRPV1），连接到可与阻遏系统同源结合的载体。以这种方式，可以将基因导入细胞，而不开始表达。只有加入强力霉素不基因开始表达，让我们来校准蛋白质expr的水平根据在生理条件所观察到的技术或水平分裂国家。我们的协议还避免了产生稳定表达的细胞系繁琐的并发症。我们首先表示从钙成像TRPV1激活的变化水平未诱导通过4小时感应以及如何在细胞内钙水平的上升密切相关的。然后，我们重复在膜片钳技术的全细胞组态的协议，表示随感应时间的增加的电流。最后，我们提出单通道电记录的例子，并且表明，基于蛋白质的个别单位寻找精确的数据收集时该技术是用于控制的表达特别有用。通过我们的协议中，我们提供了以控制在异源系统蛋白质表达的便利方式，同时避免冗长的细胞培养的并发症，因此提供了一种控制实验和提供莫之间的条件重新复制的结果。

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			<td height="21" style="height:21px;width:216px;">Doxycycline hyclate</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">D9891-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fura-2, AM ester</td>
			<td style="width:153px;">Biotium</td>
			<td style="width:119px;">BTM-50034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pluronic&#174; F-127</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">P2443-250G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#181;-Slide 8 Well</td>
			<td style="width:153px;">ibidi</td>
			<td align="right" style="width:119px;">80826</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">(E)-Capsaicin</td>
			<td style="width:153px;">Tocris</td>
			<td align="right" style="width:119px;">462</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Olympus IX70 Fluorescence Microscope</td>
			<td style="width:153px;">Olympus</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Lambda DG-4 Wavelength Switcher</td>
			<td style="width:153px;">Sutter Instruments</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EXi Blue Fluorescence Microscopy Camera</td>
			<td style="width:153px;">QImaging</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MetaFluor Fluorescence Ratio Imaging Software</td>
			<td style="width:153px;">Molecular Devices</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thin Walled Borosilicate Tubing</td>
			<td style="width:153px;">Sutter Instruments</td>
			<td style="width:119px;">B150-110-7.5HP</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Standard Walled Borosilicate Tubing</td>
			<td style="width:153px;">Sutter Instruments</td>
			<td style="width:119px;">B150-86-7.5HP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethyl sulfoxide anhydrous</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td align="right" style="width:119px;">276855</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P1000 micropipette puller</td>
			<td style="width:153px;">Sutter Instruments</td>
			<td style="width:119px;">P-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MF-900 Microforge</td>
			<td style="width:153px;">NARISHIGE</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ValveBank perfusion sysytem</td>
			<td style="width:153px;">AutoMate Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Digidata&#174; 1440A Low-noise Data Acquisition System</td>
			<td style="width:153px;">Molecular Devices</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Axopatch 200B Amplifier</td>
			<td style="width:153px;">Molecular Devices</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pCLAMP 10.6 Software</td>
			<td style="width:153px;">Molecular Devices</td>
			<td style="width:119px;">n.a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">micromanipulator</td>
			<td style="width:153px;">Sutter Instruments</td>
			<td style="width:119px;">MP-225</td>
			<td></td>
		</tr>
	</tbody>
</table>

controllable ion channel expression through inducible transient transfection

转染，外来核酸递送入细胞，是在蛋白质的研究的强有力的工具。通过这种方法，离子通道可通过电生理分析，生化表征，突变研究，以及它们对细胞过程的影响进行调查。瞬时转染提供了一个简单协议，其中蛋白质在几个小时内变得可用于分析到几天。虽然这种方法提供了一个相对简单的和时间有效的协议，关键部件中的一个被校准感兴趣的适合于分析生理相关水平或水平的基因的表达。为此目的，能够提供以控制感兴趣的基因的表达的能力的许多不同的方法已经出现。数的稳定细胞转染方案提供了一种方法以永久引入目的基因到细胞基因组中的四环素控制的转录的调控下TIONAL激活。虽然这种技术产生可靠的表达水平，每个感兴趣基因需要熟练工作包括杀灭曲线的校准，细胞集落的选择，以及整体更多的资源在几个星期。这里，我们提出使用该瞬时受体电位阳离子通道亚家族V部件1（TRPV1）基因的瞬时转染在可诱导系统来表达以受控的方式是在离子通道分析所必需的蛋白质的有效方法的协议。我们表明，使用这种技术，我们能够与具有单个转染每种类型的数据收集所需的控制信道的水平进行钙成像，全细胞，和单信道分析。总体而言，这提供了可用于研究离子通道的结构和功能可复制的技术。

快速创建一个诱导型表达模式，我们利用HEK293细胞的表达四环素阻遏蛋白（TR）（ 例如，T-REX-293），并包含在CMV启动子和多克隆位点之间的四环素操纵子序列（TETO）载体（ 例如，pcDNA4 / TO）。当转染入TREX-293细胞，在pcDNA4 / TO感兴趣的基因的表达被抑制，如TR结合TETO。添加多西环素的培养基阻止TR-TETO相互作用，从而允许转染的蛋白的表达。为了控制鼠TRPV1（rT...

Watch this Scientific Journal Video about Controllable Ion Channel Expression through Inducible Transient Transfection at JoVE.com

Controllable Ion Channel Expression through Inducible Transient Transfection

通过异源表达系统研究离子通道已成为生物医学研究核心技术。在这个手稿，我们提出了一个时间有效的方法通过诱导型启动子的控制下进行瞬时转染来实现紧密控制的离子通道的表达。

通过诱导瞬时转染可控离子通道表达

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Research

JoVE Journal

Genetics

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

科研

遗传学

The Terroir Concept Interpreted through Grape Berry Metabolomics and Transcriptomics

风土通过概念葡萄果实代谢组学和转录组解释的

Terroir refers to the combination of environmental factors that affect the characteristics of crops such as grapevine (Vitis vinifera) according to ...

Inducible T7 RNA Polymerase-mediated Multigene Expression System, pMGX

可诱导T7 RNA聚合酶介导的多基因表达系统，pMGX

Co-expression of multiple proteins is increasingly essential for synthetic biology, studying protein-protein complexes, and characterizing and harnessing ...

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

通过两个互补细胞同步方案研究细胞周期调控的基因表达

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

CRISPR/Cas9 蛋白注射到沟鲶、鮰松毛虫、基因编辑的胚胎

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

血管动力尾静脉注射对小鼠肝细胞基因的本构和诱导系统的修饰

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

昆虫细胞 (sf9) 外源基因在蛋白质功能检测中的瞬态表达

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

从土壤、根际和根 Endosphere 提取细菌群落数据的根菌群探析

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

原位杂交法在小鼠心脏切片组织特异 miRNA 表达谱分析中的应用

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

Inducible and Reversible Dominant-negative (DN) Protein Inhibition

Inducible and Reversible Dominant-negative DN Protein Inhibition

诱导和可逆显性阴性 (dn) 蛋白抑制

Dominant-negative (DN) protein inhibition is a powerful method to manipulate protein function and offers several advantages over other genome-based ...