这项工作得到了来自国家科学基金会 (NSF-PGRP-IOS-1339388) 的植物基因组研究项目的支持。

1. 植物的生长
<ol><li>母猪 5-10 大豆种子 (威廉斯 82) 在一个 13 cm 罐在温室在长的天情况下 (16 h 光在1500µmol m-2 s-1) 在25° c 在风俗土壤混合为大豆 (1:1: 1 土、珍珠岩和鱼雷砂的比值。</li>
</ol>2. 质粒 DNA 的制备
<ol><li>使用无菌吸管尖端或牙签, 选择一个单一的殖民地或冷冻甘油库存的大肠杆菌携带的质粒含有的利益基因和接种它在20毫升 Luria Bertani (LB) 液体培养基与适当的抗生素在50毫升瓶。</li>
	<li>在37° c 的瓶子上一夜之间孵育瓶。在室温下离心 1.2万 x g 的悬浮液收集细菌5分钟, 丢弃上清。按照制造商的质粒准备试剂盒的程序提取和纯化质粒。</li>
</ol>3. 原生质体分离
<ol><li>从10天的老大豆幼苗 (图 1) 中切下新扩展的单叶叶。 注: 在适宜的发育阶段选择叶片是大豆原生质体制备成功的关键。只有在早期发育阶段才使用扩大叶。随着树叶的成熟, 细胞壁变得更加难以消化。</li>
	<li>用新鲜的剃刀刀片, 从单叶叶中取出中脉, 然后将残余切成 0.5-1 毫米的条纹。 注意: 两个单叶的叶子在10毫升的酶溶液中消化, 将为超过10的转化提供足够的原生质。</li>
	<li>使用一对镊子, 立即和轻轻地转移到10毫升的新准备的酶溶液 (表 1) 在15毫升管的叶子条。真空在室温下渗透15分钟的叶子条。</li>
	<li>在室温下, 用柔和的搅拌 (40 rpm) 在低光照下孵育酶溶液中的叶子条, 4-6 小时。确保当原生质体释放时, 酶溶液变成黄绿色。检查显微镜下的酶/原生质体溶液 (X10)。 注意: 释放的原生质体是球形的, 而未消化的细胞有不规则或椭圆形的形状。</li>
	<li>将10毫升的酶/原生质体溶液在50毫升的试管中轻轻地浇注并加入10毫升的 W5 溶液 (表 1) 在室温下, 以停止消化。轻轻地倒置管几次。将酶/原生质体溶液轻轻倒入一个干净的75µm 尼龙网格上, 放在50毫升管上, 除去未消化的叶子组织。</li>
	<li>将流经的酶/原生质体溶液离心于50毫升的 100 x g, 室温下为1-2 分钟。用10毫升的血清吸管轻轻移除上清液, 不干扰原生质体颗粒。</li>
	<li>通过在显微镜下 hemacytometer 下的原生质体数 (x10), 在 W5 溶液中重原生质体并稀释到 2 x 105 mL-1的浓度。将原生质体保持在冰上30分钟。</li>
	<li>离心悬浮在 100 x g 在室温下1-2 分钟, 用1毫升吸管轻轻地移除 W5 溶液, 而不干扰原生质体颗粒。重 MMG 溶液 (表 1) 中的原生质, 在室温下浓度为 2 x 105 mL-1 。</li>
</ol>4. 原生质体转化
<ol><li>100µL 等分原生质体 (2 x 104原生质体在 2 x 105 ml-1) 在1.5 毫升低粘离心管使用未切割的200µL 吸管提示。把一个分放在一旁作为负控制。在原生质体的其余分中加入10µL 质粒 (10-20 µg)。</li>
	<li>在 1.5 mL 离心管的内壁上缓慢地加入110µL 的新制备的 PEG 溶液 (表 1), 然后轻轻地倒置和旋转管, 直到溶液变得均匀。</li>
	<li>室温下孵育转化混合物15分钟。</li>
	<li>为了停止转换, 慢慢地在室温下将400µL 的 W5 溶液加到1.5 毫升的管中, 然后轻轻地倒置管子, 直到溶液变得均匀。离心管在100克在室温下1-2 分钟, 用吸管丢弃上清液。</li>
	<li>将1毫升的 WI 解决方案 (表 1) 添加到试管和重中, 轻轻移1-2 次。添加1毫升的 5% (卷/卷) 无菌小牛血清在每个井的 6-良好的组织培养板, 以大衣的表面, 并防止原生质体黏附在板块。</li>
	<li>几秒钟后, 用吸管将小牛血清丢弃。将悬浮原生质体转移到培养板的井中。用盖子盖住盘子。</li>
</ol>5. 原生质体培养和收获
<ol><li>在室温下在黑暗中孵育原生质1-2 天。 注意: 我们发现, 两天的孵化产生更强的荧光蛋白信号一般与一天的孵化相比, 和荧光蛋白信号可能持续长达3-4 天。</li>
	<li>将原生质体溶液转移到1.5 毫升低粘离心管。离心管在 100 x g 为1-2 分钟在室温下收获原生质体。用吸管移除上清液, 并将10µL 原生质体转移到玻片上。</li>
	<li>在荧光或共聚焦显微镜下观察荧光信号。使用非转化的原生质体作为阴性控制 (没有信号应该被观察)。</li>
</ol>

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Natl. A. Sci. USA. 109 (32), E2155-E2164 (2012).</li></ol>

作者声明他们没有竞争的金融利益。

本方案对大豆原生质体的分离及在瞬态表达研究中的应用进行了全面的测试, 并在实验室中得到了很好的效果。程序简单易得, 需要普通设备和最低成本。我们的协议产生了大量的均匀, 高质量的原生质相比, 以前报告的方法34,35,36,37,38。然而, 由于有许多因素影响原生质体的?...

原生质体是细胞壁被移除的植物细胞。由于它们保持植物细胞的大部分功能和活动, 原生质体是一个良好的模型系统, 观察和评价各种细胞事件, 是有价值的工具, 研究体细胞杂交1和植物再生2。原生质体也被广泛用于植物转化3,4,5, 因为细胞壁会阻止 DNA 进入细胞。原生质体具有完整植物的一些生理反应和细胞过程, 因此为研究亚细胞蛋白定位的基础研究提供了基本的价值6,7,8,蛋白质-蛋白质相互作用9,10和启动子活动11,12,13在活细胞中。
首次报道了植物原生质体的分离, 在 1960年14 , 对原生质体分离和转化的协议进行了开发和优化。原生质体分离的标准程序包括叶片的切割和细胞壁的酶消化, 然后分离出未被消化的组织碎片释放出的原生质体。转换策略包括电穿孔15,16, 微注射17,18, 和聚乙二醇基 (PEG)4,5,19方法。广泛的物种已报告原生质体分离成功, 包括柑桔20, 芸薹21, 茄22和其他观赏植物系列23,24。虽然不同的组织类型用于各种物种, 一个系统的瞬态表达在拟南芥叶肉原生质体 (TEAMP) 分离从模型植物的叶子拟南芥已经建立良好的25并广泛应用于不同的应用程序。
大豆 (甘氨酸最大值(L.)含笑.) 是最重要的蛋白质和油料作物之一26。与拟南芥和大米不同, 获得转基因大豆植株是相当困难和低效率的。农杆菌介导的浸润已普遍用于烟草表皮细胞的瞬态基因表达研究27和幼苗在拟南芥28,29, 而农杆菌杆菌已用于大豆毛状根的转化,30。病毒诱导的基因沉默方法已被用于下调的目标基因31,32和瞬态表达式33系统的方式。原生质体为这些方法提供了一个有价值的和多才多艺的替代品。原生质体可以从大豆的地上材料中获得, 并允许快速、同步的转基因表达。然而, 自从大豆原生质体在 198334初期成功分离后, 大豆原生质体的应用有了有限的报告35、36、37、38, 主要是由于大豆原生质体的产量相对较低。
本文介绍了一种简单有效的大豆原生质体分离方法及其在瞬态基因表达研究中的应用。利用大豆幼苗的幼单叶叶, 我们能够在几个小时内获得大量的重要原生质体。此外, 我们还优化了 PEG-钙介导的转化法, 使 DNA 转化为高效的大豆原生质体是简单、低成本的。

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			<td height="21" style="height:21px;width:256px;">Jencons Powerpette Plus Pipet Controller</td>
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			<td height="24" style="height:25px;width:256px;">Zeiss 710 Confocal Microscope</td>
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			<td height="21" style="height:21px;width:256px;">LB Broth, Lennox (Powder)</td>
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			<td style="width:119px;">BP1427-500</td>
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a simple method for isolation of soybean protoplasts and application to transient gene expression analyses

大豆 (甘氨酸最大值(L.)含笑) 是一种重要的农作物品种, 已成为研究遗传和生物化学途径的豆科动物模型。因此, 建立高效的大豆瞬时基因表达系统是十分重要的。在这里, 我们报告了一个简单的协议, 大豆原生质体的制备及其应用的瞬态功能分析。我们发现, 大豆幼苗的幼单叶叶片产生了大量高质量的原生质体。通过优化 PEG-钙介导的转化方法, 利用大豆单叶原生质体实现了高转化率。该系统为检测活大豆细胞中复杂的调控和信号机制提供了一个高效、通用的模型, 有助于更好地了解豆类的不同细胞、发育和生理过程。

对10天的老大豆不同器官进行了原生质体准备 (图 1), 并在显微镜下观察了产量 (图 2)。从下胚轴和上的细胞壁几乎没有被消化, 有些细胞相互连接 (图 2B, 2C)。在子叶 (图 2D) 和根 (图 2A) 中, 单元格壁仅在单元格的一小部分中被移除。相比之下, 在使用...

Watch this Scientific Journal Video about A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses at JoVE.com

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

我们开发了一个简单和有效的协议, 以制备大量的大豆原生质体研究复杂的调控和信号机制的活细胞。

大豆原生质体分离的简易方法及其在瞬态基因表达分析中的应用

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

a-simple-method-for-isolation-soybean-protoplasts-application-to

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

24.6K 次观看.  California State University, Northridge. 我们开发了一个简单和有效的协议, 以制备大量的大豆原生质体研究复杂的调控和信号机制的活细胞。

视频: A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

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 ...

科研

生物学

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

从胚胎斑马鱼和cDNA合成的基因表达分析的RNA分离

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

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

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

嗨 - C：一个方法研究基因组的三维架构。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

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

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 to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

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 ...

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 ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

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 ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

蛋白通过流体动力基因传递在小鼠体内瞬时表达

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...