Name: analysis of the gap junction-dependent transfer of mirna with 3d-frap microscopy
Uploaded: 2017-06-19T14:00:00
Description: Watch this Scientific Journal Video about Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy at JoVE.com

德国联邦教育和研究部（FKZ 0312138A和FKZ 316159），梅克伦堡 - 西波美拉尼亚与欧盟结构基金（ESF / IVWM-B34-0030 / 10和ESF / IVBM-B35-0010 / 12），DFG（DA1296-1）和德国心脏基金会（F / 01/12）。此外，RD由罗斯托克大学医学中心（889001），DAMP基金会和BMBF（VIP + 00240）的FORUN计划支持。

根据罗斯托克大学医学中心动物护理的道德准则进行了涉及新生小鼠的本议定书中的所有步骤。 1.细胞培养皿和心肌细胞培养基的制备<ol><li>用PBS中的0.1％明胶包被细胞培养板，并在37℃下孵育4小时或在4℃下孵育过夜。取出明胶，使其在无菌层流空气流下干燥。 </li><li>准备由补充有10％胎牛血清（FBS）和1％青霉素/链霉素（P / S）的50mL的DMEM组成的细胞培养基。预热至37°C。 ...

<ol>
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<html> miRNA是细胞生理学中的关键参与者，并被证明通过使用G_s作为细胞间交换途径，作为信号分子11,12,22。目前的方案提出体外活细胞成像技术来表征使用荧光miRNA在细胞簇内的GJ依赖性穿梭。 该方案作为细胞模型系统在心肌细胞上开发。然而，如果电穿孔是miRNA转染的合适方法，则该方法可以应用于几种细胞类型。除了细胞间细胞交换，所提出的技术也适用于研究miRNA?...

小的非编码RNA是细胞基因调控的重要参与者。这些分子由与特异性靶mRNA结合的20-25个核苷酸组成，导致翻译的阻断或mRNA降解1,2 。由小RNA（例如miRNA和siRNA）进行的基因调控过程是许多不同物种中发现的高度保守的机制。特别地，miRNA分子对多种生理过程至关重要，包括增殖，分化和再生4,5 。此外，miRNA表达的失调归因于许多病理学障碍。相应地，miRNA已经被证明适合作为诊断的生物标志物和用作基因治疗的治疗剂6,7 </suP&gt;。 间隙连接（GJs）是两个相邻细胞的质膜中的专门的蛋白质结构，允许分子量高达1kD的扩散交换。已被证明对于组织发育，分化，细胞死亡和病理性疾病如癌症或心血管疾病8,9,10很重要 。已经描述了几个分子能够穿过GJ通道，包括离子，代谢物和核苷酸。有趣的是，还发现GJs为小RNA 11,12的细胞间移动提供了途径。因此，miRNA不仅可以在其产生的细胞内，而且在受体细胞内起作用。这突出了miRNA在细胞间信号转导系统中的作用。同时，数据显示该间隙连接细胞间通讯与miRNA功能密切相关。由于miRNA和GJs对组织稳态，病理学和诊断的重大影响，全面了解GJs的功能和miRNA的相关细胞间动力学有助于阐明基于miRNA的疾病的机制，并制定新的策略miRNA疗法。 根据间隙连接耦合的程度，miRNA分子在细胞间的转移可以是非常快速的过程。因此，需要一种允许可视化和量化这些调节信号分子的快速细胞间移动的方法。通常，已经应用流式细胞术和分子生物学技术来证明小RNA的穿梭11,12,13,14。然而，与FRAP微观相反复制，这些方法缺乏高时间分辨率，这是通过GJs分析miRNA的交换时强制的。此外，FRAP显微镜侵入性较小，因此代表了一种功能强大且新颖的活细胞成像技术，用于评估几种15,16,17型细胞中分子的GJ依赖性交换。 在这里，我们提出了一个详细的协议，描述了3D-FRAP应用于评估心肌细胞之间的miRNA穿梭。为此，用荧光标记的miRNA转染心肌细胞。用该miRNA标记的细胞被光漂白，并且以时间依赖的方式记录来自相邻细胞的间隙连接miRNA再流入。 FRAP实验的高时间分辨率提供了进行动力学研究以准确评估活细胞之间的miRNA和siRNA的细胞间转移的可能性。 Moreo由于小RNA可以通过具有高度不同动力学的不同机制进行交换，FRAP显微镜可以帮助澄清GJ在相应的穿梭过程18中涉及的程度。此外，3D-FRAP可用于研究GJ通透性的生理和病理学改变及其对小RNA转移的影响15,19。

<table border="0" cellpadding="0" cellspacing="0" width="698">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">neonatal NMRI mice</td>
			<td style="width:159px;">Charles River</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Gelatin</td>
			<td style="width:159px;">Sigma Aldrich</td>
			<td style="width:119px;">G7041</td>
			<td style="width:179px;">0.1% solution in PBS, sterilized</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">PBS</td>
			<td style="width:159px;">Pan Biotech</td>
			<td style="width:119px;">P04-53500</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Dulbecco&#180;s modified medium</td>
			<td style="width:159px;">Pan Biotech</td>
			<td style="width:119px;">P04-03550</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Penicillin/Streptomycin</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15140122</td>
			<td style="width:179px;">100 U/ml, 100&#181;g/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Fetal bovine serum</td>
			<td style="width:159px;">Pan Biotech</td>
			<td style="width:119px;">P30-3306</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Cell culture plastic</td>
			<td style="width:159px;">TPP</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Primary Cardiomyocyte Isolation Kit</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">88281</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">HBSS</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">88281</td>
			<td style="width:179px;">included in primary cardiomyocyte isolation kit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Trypan blue</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15250061</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:243px;">miRIDIAN Dy547 labeled microRNA Mimic&#160;</td>
			<td style="width:159px;">Dharmacon</td>
			<td style="width:119px;">CP-004500-01-05</td>
			<td style="width:179px;">dissolve in RNAse free water, stock solution 20&#181;M</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:243px;">Sodium phosphate monobasic</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">S3139</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Sodium phosphate dibasic</td>
			<td style="width:159px;">Carl Roth</td>
			<td style="width:119px;">T877.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Potassium chloride</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">P9333</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Magnesium chloride</td>
			<td style="width:159px;">Serva</td>
			<td style="width:119px;">28305.01</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Sodium succinate</td>
			<td style="width:159px;">Carl Roth</td>
			<td style="width:119px;">3195.1</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">0.05% Trypsin/EDTA solution</td>
			<td style="width:159px;">Merck</td>
			<td style="width:119px;">L2153</td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">4-well- Glass bottom chamber slides</td>
			<td style="width:159px;">IBIDI</td>
			<td style="width:119px;">80827</td>
			<td style="width:179px;">coat with 0.1% gelatin solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Amaxa Nucleofector II</td>
			<td style="width:159px;">Lonza</td>
			<td style="width:119px;"></td>
			<td style="width:179px;">program G-009 was used for Electroporation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">LSM 780 ELYRA PS.1 system</td>
			<td style="width:159px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Excel software</td>
			<td style="width:159px;">Microsoft</td>
			<td style="width:119px;"></td>
			<td style="width:179px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">&#945;-actinin antibody</td>
			<td style="width:159px;">Abcam</td>
			<td style="width:119px;">ab9465</td>
			<td style="width:179px;">dilution 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">Connexin43 antibody</td>
			<td style="width:159px;">Santa Cruz</td>
			<td style="width:119px;">sc-9059</td>
			<td style="width:179px;">dilution 1:200</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">goat anti-mouse Alexa 594 antibody</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td>A-11005</td>
			<td>dilution 1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">goat anti-rabbit Alexa 488 antibody&#160;</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td>A-11034</td>
			<td>dilution 1:300</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Connexin43 siRNA</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">AM16708 ID158724</td>
			<td>&#160;final concentration 250nM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:243px;">Near-IR Live/Dead Cell Stain Kit</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">L10119</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">CellTrace Calcein Red-Orange</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">C34851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:243px;">DAPI nuclear stain</td>
			<td style="width:159px;">Thermo Scientific</td>
			<td style="width:119px;">D1306</td>
			<td style="width:179px;"></td>
		</tr>
	</tbody>
</table>

analysis of the gap junction-dependent transfer of mirna with 3d-frap microscopy

小反义RNA（如miRNA和siRNA）在细胞生理学和病理学中起重要作用，而且可用作治疗几种疾病的治疗剂。开发miRNA / siRNA治疗新的创新策略是基于对基础机制的广泛了解。最近的数据表明，小细胞以间隙连接依赖的方式在细胞之间进行交换，从而在受体细胞中诱导基因调控作用。分子生物学技术和流式细胞仪分析通常用于研究miRNA的细胞间交换。然而，这些方法不提供高的时间分辨率，这在研究分子的间隙连接通量时是必需的。因此，为了研究miRNA / siRNA作为细胞间信号分子的影响，需要新的工具，这将允许在细胞水平上分析这些小RNA。本协议描述了ap光漂白后三维荧光恢复（3D-FRAP）显微镜的澄清，以阐明心脏细胞之间的miRNA分子的间隙连接依赖性交换。重要的是，这种直接和非侵入性的活细胞成像方法允许实时荧光标记的小RNA的间隙连接穿梭的可视化和定量，具有高的时空分辨率。通过3D-FRAP获得的数据证实了细胞间基因调控的新途径，其中小RNA作为细胞间网络内的信号分子。

在这里，我们提出3D-FRAP显微镜作为非侵入性技术研究新生儿心肌细胞荧光miRNA间隙连接穿梭。分离的心肌细胞显示典型的条纹α-肌动蛋白模式，并且沿着细胞 - 细胞边界包含大量的Cx43斑块（ 图1A ，白色箭头），这允许分子的高细胞间通量。通过显微定量α-肌动蛋白阳性细胞评估分离的心肌细胞的纯度。虽然培养物中存在一些非心肌细胞（α-肌动蛋?...

Watch this Scientific Journal Video about Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy at JoVE.com

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

在这里，我们描述了光漂白后三维荧光恢复（3D-FRAP）的应用，用于分析miRNA的间隙连接依赖穿梭。与通常应用的方法相比，3D-FRAP允许实时定量小RNA的细胞间转移，具有高的时空分辨率。

用3D-FRAP显微镜分析miRNA的间隙连接依赖性转移

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

Research

JoVE Journal

Biology

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

该<em&gt;爪蟾</em&gt;卵母细胞切割开凡士林隙电压钳技术与荧光

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

科研

生物学

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

检测miRNA靶在高通量的使用3&#39;LIFE分析

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Real-time Imaging of Plant Cell Surface Dynamics with Variable-angle Epifluorescence Microscopy

具有可变角度荧光显微镜植物细胞表面动力学的实时成像

A plant&#8217;s cell surface is its interface for perceiving environmental cues; it responds with cell biological changes such as membrane trafficking and ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

与VirusMapper超分辨率显微镜开源单粒子分析

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Biological Sample Preparation by High-pressure Freezing, Microwave-assisted Contrast Enhancement, and Minimal Resin Embedding for Volume Imaging

高压冷冻、微波辅助对比度增强和最小树脂嵌入在体积成像中的生物样品制备

The described sample preparation technique is designed to combine the best quality of ultrastructural preservation with the most suitable contrast for the ...

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

系绳双层脂膜，以监测金纳米粒子和脂膜之间的热传递

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

应用被动头部运动在啮齿动物头部产生定义的加速度

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

Measurement of Liver Stiffness Using Atomic Force Microscopy Coupled with Polarization Microscopy

使用原子力显微镜结合偏振显微镜测量肝硬度

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell ...

Fluorescence Lifetime Macro Imager for Biomedical Applications

用于生物医学应用的荧光寿命宏观成像仪

This paper presents a new photoluminescence lifetime imager designed to map the molecular oxygen (O2) concentration in different phosphorescent samples ...

Imaging of Mitochondrial Peroxide in Living Yeast Cells Using mtHyper7

Imaging of mtHyPer7, a Ratiometric Biosensor for Mitochondrial Peroxide, in Living Yeast Cells

Advancing Mitochondrial Research Â– mtHyper7 Biosensor for Subcellular Analysis (Video) | JoVE

mtHyPer7（一种用于线粒体过氧化物的比例生物传感器）在活酵母细胞中的成像

Mitochondrial dysfunction, or functional alteration, is found in many diseases and conditions, including neurodegenerative and musculoskeletal disorders, ...