1。准备和Namalwa细胞计数<ol><li>从培养瓶中取出的细胞和细胞离心沉淀。删除已知体积的PBS上清和悬浮细胞。在本议定书中所使用的细胞悬浮细胞。如果您使用的是贴壁细胞，你将需要trypsinize之前收集的。 </li><li>要确定活细胞数，染色细胞悬液等分，由0.4％台盼蓝溶液和吸管10μL混合物混合计数幻灯片上1:1，然后插入幻灯片TC10自动细胞计数器（Bio - Rad公司实验室，INC）。 </li><li> TC10细胞计数将自动对焦，以保证最精确的计数，然后使用计数进行。它会自动确定样品中的活细胞和细胞总数的密度。 </li><li>选择查看图像显示viewscreen细胞。然后选择稀释计算器TC10细胞计数，自动计算金额，将需要的细胞混合。 </li><li>本实验使用的是共有3个siRNA的对照siRNA和两个不同的siRNA针对STAT6的基因，加上未转染对照。这就要求在终浓度为5 × 10 6细胞每毫升3.6毫升。 </li><li>输入所需浓度稀释计算器和最终的音量值，并计算出细胞悬液，所需的量的基础上，样品的活细胞计数。另外，手动执行计算。 ，然后转移到一个新的管这些试验的细胞的混合物的要求量。 </li><li>旋转颗粒细胞的细胞混合物，并删除所有的PBS。重悬在3.6毫升基因脉冲电击缓冲液（Bio - Rad公司实验室）。转移到含有适当的siRNA管800μL等份，轻轻混匀。这些细胞是目前电准备。 </li></ol> 2。电穿孔细胞在96孔板<ol><li> 96电板允许复制所有四个一起被电治疗。 </li><li>一个96孔的电板适当的水井，转移到150μL的细胞悬液。 </li><li>插入MXcell电板腔板，并盖上盖子。 </li><li> Electroporate使用的细胞系，并正在使用的缓冲区的优化设置。这些细胞电穿孔使用250伏的仪器设置的指数衰减波，350μF和1000Ω电阻。 </li><li>电完成后，吸管和每口井轻轻地混合，然后将细胞转移到组织培养板中含有预热媒体。 Namalwa细胞是生长在7.5％FCS，1％丙酮酸补充的RPMI（Life Technologies公司）。 </li><li>控制尚未电穿孔的样品取自剩余的细胞悬液，并加入到预热的培养基的培养皿相同的电穿孔细胞。 </li><li>在37℃过夜孵育细胞℃，5％的CO 2。 </li><li> 24小时后，诱导细胞与10 ng / ml的终浓度重组IL - 4（R＆D系统）。控制细胞的第二组只收到其他媒体。所有的细胞都在37 ° C和5％的CO 2孵育一个额外的24小时。 </li></ol> 3。提取和检查的RNA <ol><li>共增长了48小时后，每孔计数细胞，并转移到每口井之一等份为RNA提取的离心管，并冻结了免疫印迹或其他蛋白质分析的第二个等份。 </li><li>样品离心沉淀细胞，然后取出并丢弃上清液从每管。 </li><li>继续与RNA的提取。我们通常使用的奥罗总RNA试剂盒（Bio - Rad公司）根据所包含的协议。 </li><li>当监视的siRNA介导的基因敲除，以验证RNA的质量显得尤为重要，因为降解的RNA会产生误导的结果。 260/280的比例并不表明RNA的完整性，因此重要的是要运行的样品，凝胶或微流体装置，以确认RNA的不退化。 </li><li>为了评估在一个步骤的质量和数量，分析这些样本将被使用的Experion自动电泳系统（BIO - RAD）。 </li><li>首先，热的样品。然后，总理，负载和旋涡的Experion RNA芯片。芯片插入的Experion电泳站，并开始运行。 </li><li>运行完成后，将显示的Experion软件电泳，结果表的形式（即显示RNA浓度，核糖体的比例，和RNA的质量指标数量），和虚拟凝胶结果自动（看起来类似传统的RNA凝胶）。高质量的总RNA样本，通常会有8-10 RQI。 </李&gt; </ol> 4。实时PCR <ol><li> RNA质量核实后，进行cDNA的反应。本实验所用的iScript一步法RT - PCR混合物（BIO - RAD）组合在一个反应​​合成cDNA和实时PCR。 </li><li>设为主结构，包含每个引物RNA模板以外的所有反应组件，并分发到PCR板。 </li><li>一个统一的浓度稀释后的RNA，RNA样品添加适当的水井。每一个反应是成立一式三份。 </li><li>封板，并放入的CFX384实时PCR系统（BIO - RAD）。选择合适的协议，并开始运行。 </li><li>一旦运行完成后，基因表达是通过比较基因表达的基因归参照基因在细胞电穿孔与实验的siRNA细胞电穿孔与对照siRNA正常化的利息确定。 </li></ol> 5。的siRNA敲除分析<ol><li>使用实时定量PCR监测调查中IL - 4 CCL17依赖诱导转录激活因子的作用STAT6的表达CCL17。在这个实验中，被用作参考基因GAPDH的。备用参考基因​​或多个参考基因可以用相同的方式。 </li><li> CFX Manager软件会自动归CCL17表达从同一样品的参考基因表达，并自动比较造成的标准化值，以控制通过简单的选择GAPDH作为“参考”和控制治疗（IL - 4诱导的无控制的siRNA）的治疗CFX Manager软件在实验中设置窗口的“控制”。 </li><li>无IL - 4诱导CCL17的转录低，构水平保持在控制治疗相媲美。 </li><li>诱导的IL - 4和保留正常STAT6通路（即，对照siRNA或未转染对照处理）的细胞表现出8-10倍CCL17表达诱导。 </li><li>与IL - 4，但与STAT6的表达，通过引进任STAT6的siRNA抑制细胞显示，只有约2倍高于非诱导细胞在IL - 4诱导表达的CCL17确认的STAT6作用CCL17表达。 </li></ol> 6。代表性的成果<img src="/files/ftp_upload/1904/1904fig1.jpg" alt="图1" /> 图1。 IL - 4信号转导通路的概述 。 的IL - 4及其受体）结合，导致二聚和STAT6的活化。激活STAT6区转移到细胞核，并激活其靶基因，如CC17，转录。 b）在不IL - 4诱导CCL17的转录将保持在低，构水平。 c）在STAT6基因的siRNA介导的沉默，与IL - 4的治疗应该导致很少或根本没有增加CCL17表达。 <img src="/files/ftp_upload/1904/1904fig2.jpg" alt="图2" /> 图2。由IL - 4诱导CCL17测量不同条件下，通过定量实时PCR 。 一）种植无IL - 4的样本低构的表达水平，不论基因的表达。 B）在绿色和蓝色所示，对照样品的反应通常与IL - 4治疗; 8-10倍CCL17诱导。然而，针对STAT6的，在红色和粉色的siRNA转染的细胞，减少了CCL17表达IL - 4，支持这个想法，STAT6起着作用的IL - 4诱导表达的CCL17。

笔者采用Bio - Rad公司实验室产生本文中使用的试剂和仪器。

本文演示了如何electroporate陷入困境的siRNA转染悬浮细胞系，如何遵循这些细胞的基因表达。执行此过程时，重要的是要记得保持尽可能一致的，所有样品的事情。

using an automated cell counter to simplify gene expression studies: sirna knockdown of il-4 dependent gene expression in namalwa cells

使用的siRNA介导的基因敲除继续在基因表达研究中的一个重要工具。 siRNA的研究，不仅要研究单个基因的下调的影响，但也询问信号通路和其他复杂的相互作用网络。这些途径分析需要使用有关的细胞模型和方法，导致少扰动细胞生理学。电穿孔是越来越多地被用来作为一种有效的方式引入难以siRNA和其他核酸，细胞株和原代细胞的转染而不改变正在调查中的信号转导通路。有一个成功的siRNA实验的多个关键步骤，并且有办法在几个实验阶段，同时提高数据的质量，简化工作。为了帮助您与您的siRNA介导的基因敲除项目开始，我们将演示如何执行一个途径的研究，从收集和计数细胞前通过后转实时PCR基因表达分析，以电的完整。下面的研究探讨IL - 4在Burkitt淋巴瘤细胞系（Namalwa）CCL17依赖基因表达的转录激活因子STAT6的作用。本文演示的技术是很有用的一个广泛的siRNA实验的基础上壁和悬浮细胞。我们还将展示如何精简细胞与TC10自动细胞计数计数，如何electroporate MXcell电系统同时使用多个样品，以及如何同时评估RNA的质量和数量的Experion自动电泳系统。

Watch this Scientific Journal Video about Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells at JoVE.com

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

此过程介绍了一个快速简便的工作流程，引入困难的siRNA转染细胞系，并按照实时PCR基因表达。使用自动细胞计数器，多井的电板，全自动电泳站提供快速和可靠的结果，而不需要昂贵的机械手。

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

using-an-automated-cell-counter-to-simplify-gene-expression-studies

Research

JoVE Journal

Biology

15.5K 次观看.  Bio-Rad Laboratories.  中文 

视频: Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

Bacterial Gene Expression Analysis Using Microarrays

使用微阵列的细菌基因表达分析

科研

生物学

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

脊髓电

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

机器人技术和植物组织的基因表达研究的动态图像分析

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9.
The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.

E4orf4诱导的细胞死亡的击倒效果的基因表达和测量条件击倒细胞系的制备

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

siRNA的筛选识别泛素和泛素样系统压力表生物学途径在培养的哺乳动物细胞

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...