We thank Lowell Cochran for expert videography assistance. The financial assistance of the Canadian Institutes of Health Research (CIHR), the Canadian Breast Cancer Foundation (CBCF, Ontario Chapter), the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Breast Cancer Research Alliance, the Ontario Centers of Excellence, the Breast Cancer Action Kingston and the Clare Nelson bequest fund through grants to LR is gratefully acknowledged. SG was the recipient of an NSERC studentship. MG was supported by a postdoctoral fellowship from the US Army Breast Cancer Program, the Ministry of Research and Innovation of the Province of Ontario and the Advisory Research Committee of Queen&#8217;s University.

1，电镀的电穿孔室中的电解槽<ol><li>在层流罩，使用无菌技术如常trypsinize细胞。 注意：通过离心来消除胰蛋白酶的所有痕迹，因为它们可能会妨碍细胞的玻璃板上散布，粘着和间隙连接从而形成是非常重要的。 </li><li>吸管将1ml细胞悬液中提供的原位电穿孔仪（ 图1），并将其放置在37℃， 二氧化碳培养箱无菌电室的。 注：细胞粘附可以通过电镀于纤连蛋白，胶原蛋白，多聚赖氨酸或细胞和组织粘合剂（见材料表）而得到提高。 </li><li>当细胞已经形成了融合层，他们已经准备好细胞间隙连接通讯检查。 </li></ol> 2，电穿孔程序电穿孔的细胞间隙连接通讯检查可以在层流罩外进行，因为只需要几分钟的时间。如果潜伏期较长时间都需要一个特定的实验，那么就可以完全在层流罩进行。在所有情况下，当细胞生长腔室必须是无菌的。 <ol><li>制备5毫克/毫升荧光黄液：将10毫克LY粉末（设置有电穿孔仪）在2ml无钙生长培养基中。对于实验中，需要较长的培育时间，过滤消毒溶液中，并储存在4℃。 </li><li>吸出培养基，用无钙培养基洗细胞，小心不要划伤单层或干细胞。如果细胞是干他们通常有较深的细胞核，并拿起LY没有电。的效果是在它更多地暴露于空气气流（ 图4F）滑动的中间更加明显。 </li><li>用Eppendorf移液器，吸管的染料溶液到细胞（400μl的整个腔室），在该腔室，b的边缘eing小心不要触摸细胞层。 </li><li>放置室与电穿孔仪附带的支架，并应用适当的强度（见讨论）的脉冲。 </li><li>小心吸一些LY解​​决方案。它可以重复使用的第二时间不太重要的实验。 </li><li>加入含10％透析血清钙的DMEM。 </li><li>孵育的细胞为3-5分钟，在培养箱中，以允许通过间隙连接的染料转移。 注：在这一点上列入透析血清帮助毛孔关闭。 </li><li>与钙的DMEM活细胞观察洗未掺入的染料。备选地，细胞可以固定在该阶段，通过加入4％甲醛的井，然后用PBS（磷酸盐缓冲盐水）。在所有情况下，清洗必须移除所有背景。 注：在初步实验，以确定最佳的条件下，如果电压过低，则有可能让细胞恢复在incubator表示了几分钟，电穿孔的同一张幻灯片第二次来保存幻灯片的成本。 </li></ol> 3，显微镜检查<ol><li>观察细胞的荧光下照射，使用倒置显微镜配备有适当的过滤器的染料被使用。对于荧光黄，激励是423nm，发射555nm处，全身振动过滤器尼康IX70显微镜。 </li><li>消除液面效应通过放置一个玻璃盖玻片上的塑料井的顶部，它填充到液体上方后，检查下相衬细胞形态。为更好的画面，以及可从滑动被分离，并在盖玻片放置在框架上。对于固定的细胞中，以及可填充有甘油的显微镜观察，而对于活细胞就必须充满生长培养基中。 注意：洗涤和拍摄是比较容易，如果下列染料（上述步骤2.7）的转移将细胞用甲醛固定。如果细胞是无吨固定，荧光从细胞取决于LY的介绍，而在固定的细胞的荧光被保持几个小时的细胞类型，电压和量消除内约60分钟。然而，荧光消失或O 2 / N孵化后消散，即便是在固定的细胞。出于这个原因，照片必须尽快电穿孔（ 图2）后采取。 </li></ol> 4，定量间通讯<ol><li>拍摄的细胞在荧光下，相位相反（见图3）20倍的目标。 </li><li>识别并用星标记电 ​​穿孔的细胞在边界与非电穿孔的区域（ 图2中 ，箭头，电穿孔边缘）。 </li><li>识别并以点标记的荧光细胞上的非电穿孔的一侧，其中所述染料已通过间隙连接的传输（ 图3A和3B）。 </li><li>除以总数通过电穿孔的细胞沿着边缘（ 图3A和3B）的数量的荧光细胞上的非电穿孔区域。 请注意：从至少200个连续的电穿孔的边缘细胞的转移计算每个实验。得到的数字是GJIC值。 </li></ol>

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The corresponding author is the inventor in a patent held by Queen&#8217;s University, which has been licensed to Cell Projects Inc. The company or the University had no influence upon the contents of the paper.

在协议中的关键步骤 电穿孔材料 该材料的纯度被电穿孔是非常重要的。如果细胞是非常平坦的，则高浓度的示踪染料必须使用（不超过20毫克/毫升为LY），并在这种情况下，纯度，甚至比在细胞有更多的球状1更为重要。除LY大量的各种其它染料或nonpermeant分子已被采用，例如为一系列的Alexa染料作为探针的通道由不同的连接蛋白25的使用。然而?...

电电流的一个小区中的应用程序会导致孔的细胞膜上形成，由一个过程被称为电穿孔。的孔允许的各种透过膜nonpermeant分子的通道。电场可以精确地控制，从而使形成的孔是非常小的和重新闭合迅速，以最小的干扰对细胞生理学。有趣的是，贴壁细胞可生长在涂有导电性和透明的铟锡氧化物（ITO）和电穿孔的原位载玻片上，即在那里生长的表面上，而不被分离的悬浮液中进行电穿孔。细胞可以生长得非常好在该表面上，并且当它们被安装并伸展，详细显微镜观察是可能的。使用这种技术，小型nonpermeant分子可即刻导入和成基本上100％的细胞，这使得该技术特别适用于研究排版的活化通路的继受体（1审查）的配体刺激onents。 电穿孔已经大多用于引入的DNA（也称为electrotransfection）。然而，电穿孔法在原位可用于引入了大量的各种分子，如肽2-4，寡核苷酸，如反义RNA，双链DNA诱饵寡核苷酸的宝贵抑制转录因子的结合，或siRNA 3,5， 6，放射性7-10个核苷酸，蛋白质11 12或前体药物13。在电穿孔之后，将细胞可被裂解为生化分析或固定和染色的抗体。 导电性ITO涂层非常薄，800-1,000Å，使细胞的生长不是由在两个表面的高度差的干扰，如在单元两端的导电涂层的边缘生长。这提供的优点是，非electroporated细胞可以生长并排电穿孔者，作为对照。同样的方法可以用于间隙连接，细胞间通讯（GJIC）的检查，如在视频中描述。 间隙连接是连接相邻的电池14的内部通道。间隙连接，细胞间通讯在肿瘤形成和转移中起重要作用，而癌基因如Src蛋白抑制GJIC 15,16。审查GJIC荧光染料如荧光黄（LY）经常被导入培养的细胞中，通过显微注射或刮除装17和染料扩散到邻近细胞下的荧光照明显微观察。这些技术不过总是导致细胞损伤。我们现在描述那里的细胞生长在其上部分涂有ITO 18载玻片的技术。电脉冲在LY（或其它染料）的存在下施加连载ca利用其渗透到细胞上滑动的导电部分越来越大，而染料迁移到相邻的，非电穿孔的细胞通过荧光照明显微观察。为了避免干扰敏感的细胞可能离开单层分离，被设计的组件，其并不需要一个电极被放置在小区的顶部施加的电流19。这种方法提供了定量GJIC在大量细胞的能力，而没有任何可检测的干扰细胞代谢，由下列血清刺激12没有在G1期的长度的影响如所示，增加在fos基因的水平原癌基因蛋白（Raptis，未发表）或两个与细胞应激时，p38的生猪或JNK / SAPK 激酶相关激酶。这种方法成为可能的癌基因的表达，转化和GJ的水平之间的关系的检查集成电路18，以及Src和Stat3的的后GJIC在多种细胞类型中，包括细胞肺肿瘤样本20-23新鲜培养的效果。此外， 原位电穿孔与所描述的设置，其中缺乏的顶电极已被成功地使用，用于在脂肪细胞分化的间隙连接闭合的示范，虽然细胞附着到基底减小在该阶段19,24。

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			<td height="42" style="height:42px;width:179px;">Name of Material/ Equipment</td>
			<td style="width:428px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:179px;">DMEM</td>
			<td style="width:428px;">Cellgro http://www.cellgro.com/</td>
			<td style="width:119px;">50-013-PB</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:179px;">DMEM without Calcium</td>
			<td style="width:428px;">Hyclone (Thermo scientific: http://www.thermoscientific.com)</td>
			<td style="width:119px;">SH30319-01</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Donor Calf Serum&#160;</td>
			<td style="width:428px;">PAA: http://www.paa.com/</td>
			<td style="width:119px;">Cat.# B15-008</td>
			<td></td>
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			<td height="21" style="height:21px;width:179px;">Fetal Bovine Serum</td>
			<td style="width:428px;">PAA: http://www.paa.com/</td>
			<td style="width:119px;">Cat.# A15-751</td>
			<td></td>
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			<td height="21" style="height:21px;width:179px;">Electroporation apparatus</td>
			<td style="width:428px;">Cell Projects Ltd UK: http://www.cellprojects.com/</td>
			<td style="width:119px;">ACE-100</td>
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			<td height="21" style="height:21px;width:179px;">Chambers</td>
			<td style="width:428px;">Cell Projects Ltd UK</td>
			<td style="width:119px;">ACE-04-CC</td>
			<td>4-wells</td>
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			<td height="21" style="height:21px;width:179px;">Chambers</td>
			<td style="width:428px;">Cell Projects Ltd UK</td>
			<td style="width:119px;">ACE-08-CC</td>
			<td>8-wells</td>
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			<td height="21" style="height:21px;width:179px;">Lucifer Yellow</td>
			<td style="width:428px;">Cell Projects Ltd UK</td>
			<td style="width:119px;">ACE-25-LY</td>
			<td>High purity</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:179px;">CelTak</td>
			<td style="width:428px;">BD Biosciences</td>
			<td style="width:119px;">354240</td>
			<td>Cell and tissue adhesive</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Fibronectin</td>
			<td style="width:428px;">Sigma Aldrich</td>
			<td style="width:119px;">F1141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Collagen</td>
			<td style="width:428px;">BD Biosciences</td>
			<td style="width:119px;">354236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:179px;">Poly-Lysine</td>
			<td style="width:428px;">Sigma Aldrich</td>
			<td style="width:119px;">P8920</td>
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a functional assay for gap junctional examination; electroporation of adherent cells on indium-tin oxide

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. A variety of molecules, such as peptides or oligonucleotides can be introduced into essentially 100% of the cells in a non-traumatic manner.&#160; Here, we describe how it can be used to study intercellular, gap junctional communication. Lucifer yellow penetrates into the cells when an electric pulse, applied to the conductive surface on which they are growing, causes pores to form through the cell membrane. This is electroporation. Cells growing on the nonconductive&#160;glass surface immediately adjacent to the electroporated region do not take up Lucifer yellow by electroporation but do acquire the fluorescent dye as it is passed to them via gap junctions that link them to the electroporated cells. The results of the transfer of dye from cell to cell can be observed microscopically under fluorescence illumination. This technique allows for precise quantitation of gap junctional communication. In addition, it can be used for the introduction of peptides or other non-permeant molecules, and the transfer of small electroporated peptides via gap junctions to inhibit the signal in the adjacent, non-electroporated cells is a powerful demonstration of signal inhibition.

图2显示了大鼠肝脏上皮T51B细胞22，在荧光下（图A和B），电穿孔和LY和拍照，或相差（C）的照明，以下定影和水洗。在图A中，将电穿孔的区域的边缘被标记为红色。荧光的红色线右边的梯度表示通过间隙连接的传输。在图3A和图3B中 ，间隙连接通讯的定量表示：将细胞在电穿孔的区域的边缘被确定，并用星号标记，而其中的染料转移了细胞被标记为?...

Watch this Scientific Journal Video about A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide at JoVE.com

A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

对缝隙连接检查功能测定;贴壁细胞的氧化铟锡电

In this technique, cells are cultured on a glass slide that is partly coated with indium-tin oxide (ITO), a transparent, electrically conductive material. ...

a-functional-assay-for-gap-junctional-examination-electroporation

Research

JoVE Journal

Biology

9.8K 次观看.  Queen's University. This presentation demonstrates a method whereby electroporation of adherent, cultured cells is used for the study of intercellular, junctional communication, while the cells grow on a slide coated with conductive and transparent indium-tin oxide.

视频: A Functional Assay for Gap Junctional Examination; Electroporation of Adherent Cells on Indium-Tin Oxide

Patterning Cells on Optically Transparent Indium Tin Oxide Electrodes

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular constructs serving as in vitro mimics of native tissues.  In this study, photolithography and wet etching techniques were used to fabricate individually addressable indium tin oxide (ITO) electrodes on glass substrates.  The glass substrates containing ITO microelectrodes were modified with poly(ethylene glycol) (PEG) silane to make them protein and cell resistive.  Presence of insulating PEG molecules on the electrode surface was verified by cyclic voltammetry employing potassium ferricyanide as a redox reporter molecule.  Importantly, the application of reductive potential caused desorption of the PEG layer, resulting in regeneration of the conductive electrode surface and appearance of typical ferricyanide redox peaks.    Application of reductive potential also corresponded to switching of ITO electrode properties from cell non-adhesive to cell-adhesive. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.  Micropatterning of several cell types was demonstrated on these substrates.   In the future, the control of the biointerfacial properties afforded by this method will allow to engineer cellular microenvironments through the assembly of three or more cell types into a precise geometric configuration on an optically transparent substrate.

Non-fouling PEG silane monolayer was desorbed from individually addressable ITO electrodes on glass by application of a reductive potential. Electrochemical stripping of PEG-silane layer from ITO microelectrodes allowed for cell adhesion to take place in a spatially defined fashion, with cellular patterns corresponding closely to electrode patterns.

在光学透明的铟锡氧化物电极的图案细胞

The ability to exercise precise spatial and temporal control over cell-surface interactions is an important prerequisite to the assembly of multi-cellular ...

科研

生物学

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

氧化铁标记的胚胎干细胞和的hMSCs纳米粒子在体内的跟踪与非侵入性磁共振成像

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

在子宫内和前体内的基因表达在小鼠视网膜神经节细胞的电穿孔

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

原位贴壁和非贴壁的哺乳动物细胞的亚细胞分馏

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

克隆形成含量：贴壁细胞

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

模仿人类小分子RNA的LightSwitch萤光素酶检测系统使用3&#39;UTR记者构造的microRNA识别目标在贴壁细胞

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific shapes. Drosophila tracheal terminal cells have become a powerful genetic model to identify and elucidate the roles of genes required for generating cellular morphologies. Terminal cells are a component of a branched tubular network, the tracheal system that functions to supply oxygen to internal tissues. Terminal cells are an excellent model for investigating questions of cell shape as they possess two distinct cellular architectures. First, terminal cells have an elaborate branched morphology, similar to complex neurons; second, terminal cell branches are formed as thin tubes and contain a membrane-bound intracellular lumen. Quantitative analysis of terminal cell branch number, branch organization and individual branch shape, can be used to provide information about the role of specific genetic mechanisms in the making of a branched cell. Analysis of tube formation in these cells can reveal conserved mechanisms of tubulogenesis common to other tubular networks, such as the vertebrate vasculature. Here we describe techniques that can be used to rapidly fix, image, and analyze both branching patterns and tube formation in terminal cells within Drosophila larvae. These techniques can be used to analyze terminal cells in wild-type and mutant animals, or genetic mosaics. Because of the high efficiency of this protocol, it is also well suited for genetic, RNAi-based, or drug screens in the Drosophila tracheal system.

Examination of Drosophila Larval Tracheal Terminal Cells by Light Microscopy

Here, we present a method for light microscopy analysis of tracheal terminal cells in Drosophila larvae. This method allows for quick examination of branch and lumen morphology in whole animals and would be useful for analysis of individual mutants or screens for mutations affecting terminal cell development.

考试<em&gt;果蝇</em幼虫气管终端细胞光镜

Cell shape is critical for cell function. However, despite the importance of cell morphology, little is known about how individual cells generate specific ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

测量外产酸和分析，以确定糖酵解率

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...