benthamiana烟草植物生长温度控制在25 ° C时生长室。使用3-6周老植物叶片完全展开的健康。 提示:通过使用新兴的叶子获得更好的成果 1。农杆菌介导的瞬态渗透协议: 第1天<ol><li>农杆菌介导的基因（S）来检测细胞死亡和载体，含有适当的载体（应变农杆菌）的甘油库存数的LB /利福平（25微克/毫升）/卡那霉素（100微克/毫升）的琼脂平板构推下的GUS的录像带。始终包括空载体作为阴性对照的控制。 </li><li>孵育28 ° C为2天。 </li></ol>第3天<ol><li>接种2毫升/利福平/卡那霉素板以前挑染LB从每个单菌落含利福平（25微克/毫升）和卡那霉素（100μg/ ml的）的LB。 </li><li>孵育晃动在28 ° C直到24小时在200转达到最大生长密度是每一种文化。 </li></ol> 提示:结块，有时观察，在这种情况下，文化需要彻底的重新悬浮在出发前（即上下吹打起来） 。 第4天<ol><li>潜伏期之后，增加的总体积至10 mL新鲜的LB（含适当的抗生素）和乙酰丁香酮（终浓度为25微米）。 </li><li>另外16小时在28 ° C摇晃孵育每一种文化。 </li></ol>第5天<ol><li>以下一天洗两次每一种文化在4000 XG在室温为10分钟，加入10毫升（不添加乙酰丁香酮）渗透介质（10毫米硫酸镁4 0.7 H 2 O，9毫米的MES，pH值5.6）和离心机。 </li><li>最后的离心步骤后，重悬在5毫升渗透介质中含有乙酰丁香酮（100微米）的每一种文化。 </li><li>测量OD 600nm处 ，测量0.1和0.9之间是可以接受的的。 提示:低至0.1 OD 600nm处有没有问题，但是，更高的消耗臭氧层物质可能会产生更一致的结果。 </li><li>孵育3个多小时的文化准备农杆菌感染的文化。 </li><li>混合文化包含的基因（S）进行分析和负控制在1:1与文化包含的GUS盒的比例（含空载体的农杆菌介）。 </li><li>渗透的新兴使用1毫升的针头的注射器（图1）叶的背面（下）方。 提示:渗透阴性对照的混合物（空载体+ GUS的卡带）和包含要检测基因（基因X + GUS的卡带）对面的同种植物叶的混合物 。 每处理一式三份植物。 </li><li> 3天渗透后消费渗入叶片GUS蛋白表达和检测。 </li></ol> 2。组织化学GUS分析第8天<ol><li>真空渗入切除叶的X - gluc基板介质。 </li><li>在黑暗中孵育，室温过夜，直到出现明显的蓝染色。 </li></ol>第9天<ol><li> distillated水冲洗。 </li><li>在70％的乙醇孵育直到叶绿素被删除，然后再次转移到蒸馏水。目视评估GUS基因表达水平。 </li></ol> 3。荧光杯测定: 第8天<ol><li>隔离磨在使用液氮的砂浆渗入叶圆片总蛋白。新增100 UL GUS提取缓冲液（50毫米NAPI pH值7.0，10 mM的EDTA，0.1％TRITON X - 100，0.1％的N - lauroylsarcosine钠盐，β-巯基乙醇（0.7μL/毫升），同时保持冰样本。 </li><li>在14000克离心悬浮液在4 ° C 5分钟，取上清液可溶性总蛋白。 </li><li>根据制造商的指示使用nanodrop测量浓度。 </li><li>调整蛋白浓度到100微克每个使用GUS提取缓冲液的样品的总可溶性蛋白的。 </li><li>添加杯（4 methylumbelliferyl -β- D -葡糖苷酸三水）准备GUS提取缓冲液中终浓度为2毫米的基板。每个样品100μL（蛋白质+杯基板）的总量应该足够了。 </li><li> 1小时的反应在37 ° C。 </li><li>加入800μLGUS终止缓冲液（0.2 M NA 2 CO 3）停止反应。 </li><li>测量荧光激发波长为365 nm和发射波长为455纳米和穆标准曲线比较值的一个使用酶标仪。 GUS活性的报告nmol MU /μg总可溶性蛋白/分钟的水平。 </li></ol> 4。股票和解决方案: <ol><li>乙酰丁香酮（1米）股票（FW = 196.2克） </子&gt; - 0.981克5毫升的DMSO </li><li>卡那霉素（100毫克/毫升）的股票-准备在卫生署2 0 -过滤消毒</li><li>利福平（25毫克/毫升）的股票 - 准备在DMSO </li><li>卢里亚Bertani（LB）液体生长介质:（1L） 1％（W / V）细菌用胰蛋白胨，10克 细菌用的酵母提取物0.5％（W / V），5克 170毫米，氯化钠10克 pH值 - 7 </li><li>乙酰丁香酮（25μM）= 1米的股票最终体积为1 mL 25μL </li><li>渗透介质:（1L） 10毫米硫酸镁4 0.7 DH 2 0（FW = 246.48克），2.4648克 9毫米MES（FW = 213.25克），1.91925克 pH值 - 5.6 </li><li>乙酰丁香酮（100μM）= 100μL一个1M股票，最终体积为1 mL </li><li> GUS提取缓冲液 50 mM的那匹pH值7.0，10 mM的EDTA，0.1％TRITON X - 100，0.1％的N - lauroylsarcosine钠盐，β-巯基乙醇（0.7μL/毫升） </li><li> 2X磷酸盐缓冲液 0.2M的NaH 2 PO 4和0.2 M娜2 HPO 4 pH值7 </li><li> X - Gluc底物溶液。 溶解1毫克的5 - 溴-4 - 氯-3 - 吲哚BD -葡糖苷酸（X - Gluc）0.1毫升甲醇。添加1毫升2X磷酸缓冲液，20微升0.1M铁氰化钾，加入10μlTRITON X - 100的10％和850μLdistillated水。 </li></ol> 5。代表性的成果: 根据这一协议，GUS表达的损失预计时发生细胞死亡。我们同时表示，记者的基因，与细胞质保护拟南芥BCL - 2相关athanoGene（袋）系列的成员β-葡萄糖醛酸酶（GUS ）的。我们的合作渗透北路benthamiana离开一个35S驱动包表达盒和使用农杆菌菌株LBA4404 GUS载体pCAMBIA 2301。正如图2所示，在GUS染色明显增加之后，观察到这种渗透。相反，当已知的促凋亡的Bcl - 2家族成员伯灵顿使用，被观察的GUS染色明显减少（图2）。在这两种情况下，GUS表达比对照明显不同。然而，在表达上的差异不太明显时，荧光杯检测可以进行quanitate GUS表达。 <img src="/files/ftp_upload/2680/2680fig1.jpg" alt="图1"/> 图1。 农杆菌N.背面一侧的混合物渗透的范例benthamiana叶使用1毫升的针的注射器。 <img src="/files/ftp_upload/2680/2680fig2.jpg" alt="图2"/> 图2。一个GUS载体，共同表达在 N benthamiana叶与反死亡的基因，称为诱导细胞死亡。 GUS的水平相比，一个空向量控制（控制的GUS）。

<ol>
	<li>Jefferson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GUS+fusions:+,+&#946;-glucuronidase+as+a+sensitive+and+versatile+gene+fusion+marker+in+higher+plants.">GUS fusions: , &#946;-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.</a> EMBO J. 6, 3901-3901 (1987).</li><li>Nishihara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+&#946;-Glucuronidase+Gene+in+Pollen+of+Lily+(Lilium+longiflorum),+Tobacco+(Nicotiana+tabacum),+Nicotiana+rustica,+and+Peony+(Paeonia+lactiflora)+by+Particle+Bombardment.">Expression of the &#946;-Glucuronidase Gene in Pollen of Lily (Lilium longiflorum), Tobacco (Nicotiana tabacum), Nicotiana rustica, and Peony (Paeonia lactiflora) by Particle Bombardment.</a> Plant Physiol. 102, 357-357 (1993).</li><li>Hodal, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Science. 87, 115-115 (1992).</li><li>Kabbage, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BAG+proteins:+a+ubiquitous+family+of+chaperone+regulators.">The BAG proteins: a ubiquitous family of chaperone regulators.</a> Cell Mol. Life Sci. . 65, 1390-1390 (2008).</li><li>Kang, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AtBAG6,+a+novel+calmodulin-binding+protein,+induces+programmed+cell+death+in+yeast+and+plants.">AtBAG6, a novel calmodulin-binding protein, induces programmed cell death in yeast and plants.</a> Cell Death Differ. 13 (1), 84-84 (2006).</li><li>Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Science. 165 (1), 1-1 (2006).</li><li>Takayama, S., Reed, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+chaperone+targeting+and+regulation+by+BAG+family+proteins.">Molecular chaperone targeting and regulation by BAG family proteins.</a> Nat Cell Biol. 3 (10), 237-237 (2001).</li><li>Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+functional+analysis+of+BAG-1:+a+novel+Bcl-2-binding+protein+with+anti-cell+death+activity.">Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity.</a> Cell. 80 (2), 279-279 (1995).</li><li>Vitha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+&#946;-glucuronidase+assay+in+transgenic+plants.">Quantitative &#946;-glucuronidase assay in transgenic plants.</a> Biol. Plant. 35, 151-151 (1993).</li><li>Otha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+Expression+in+Tobacco+of+a+&#946;-Glucuronidase+(GUS)+Reporter+Gene+Containing+an+Intron+Within+the+Coding+Sequence.">Construction and Expression in Tobacco of a &#946;-Glucuronidase (GUS) Reporter Gene Containing an Intron Within the Coding Sequence.</a> Plant Cell Physiol. 31, 805-805 (1990).</li></ol>

<p class="jove_content"它往往是很难使用在哺乳动物系统中常见的植物细胞凋亡检测技术。 GUS报告制度相结合，我们目前的植物为基础的，敏感的方法检测和分析细胞死亡的球员。这种方法需要利用简单的事实，活细胞都需要GUS基因表达发生。为了确保测试结果有意义和可重复性，它是窝藏GUS卡带和要检测的基因，文化渗透在同等比例的关键。这些比率应保持比较时，额外的潜在细胞死亡的球员。如上所示，我们已经?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>利福平</td><td> VWR </td><td> IC19549001 </td><td> 25mg/mL股票在DMSO </td></tr><tr><td> 4&#39; -羟基-3&#39;，5&#39; -二甲（乙酰丁香酮） </td><td> VWR </td><td> TCD2666 </td><td> 0.981克/毫升（1M股票）在DMSO </td></tr><tr><td> 2 - （4 - 吗啉）乙磺酸一水（MES） </td><td> VWR </td><td> EM - 6110 </td><td> 1.92克/ L水为1升的渗透媒体</td></tr><tr><td>细菌用的蛋白胨</td><td>费舍尔</td><td> BP1421 - 2 </td><td> 10G / L水1 L LB培养基</td></tr><tr><td>细菌用的酵母提取物</td><td> VWR </td><td> EM1.03753.0500 </td><td> 5克/ L水1 L LB培养基</td></tr><tr><td>氯化钠</td><td> VWR </td><td> EM - 7710 </td><td> 10G / L水1 L LB培养基</td></tr><tr><td>钠磷酸二氢水合物</td><td> VWR </td><td> MK - 7868 - 12 </td><td> 2.5克/ L的水缓冲区NAPI 50MM </td></tr><tr><td>钠磷酸氢二钠亚铁</td><td> VWR </td><td> EMD - SX0715 - 1 </td><td> 5克/ L水为50mm的缓冲NAPI </td></tr><tr><td>七水硫酸镁</td><td> VWR </td><td> EM - MX0070 - 1 </td><td>为2.5 g / L水为1升的渗透媒体</td></tr><tr><td> N - Lauroylsarcosine </td><td> VWR </td><td> TCL0151 - 500G </td><td> 0.1％V / V GUS缓冲</td></tr><tr><td> 5 - 溴4 - 氯-3 - indoxyl -β- D -葡糖苷酸cyclohexylammonium盐（X - gluc） </td><td>黄金生物技术</td><td> G1281C </td><td> 1mg/100uL在甲醇100％ </td></tr><tr><td> 4 Methylumbelliferyl -μ- D -葡糖苷酸水合物（杯） </td><td>西格玛</td><td> M5664 </td><td> 2毫米，100 UL GUS提取缓冲液</td></tr><tr><td>钾亚铁三水</td><td> VWR </td><td> EM - PX1460 - 1 </td><td> 100mm的股票为X - gluc底物溶液</td></tr><tr><td> β- Methylumbelliferone（亩） </td><td> Sigma - Aldrich公司</td><td> M1381 </td><td>对于万亩标准曲线使用GUS提取缓冲液</td></tr><tr><td>碳酸钠</td><td> VWR </td><td> EM - SX0395 - 11 </td><td> 0.2米的水使GUS停止缓冲</td></tr></tbody></table>

a &#946;-glucuronidase (gus) based cell death assay

我们已经开发出一种新的瞬态植物表达系统，同时表达报告基因β-葡萄糖醛酸酶（GUS）的细胞死亡与公认的积极或消极的监管机构，。在这个系统中，N benthamiana叶渗透与35S驱动的表达，其中包含要分析的基因磁带，GUS载体pCAMBIA 2301使用车辆农杆菌菌株LBA4404。由于活细胞所需的GUS基因表达发生，GUS活性的损失预计时，这个标记基因与细胞死亡的积极监管机构的合作表示。同样，增加的GUS活性是观察时使用抗凋亡基因相比，矢量控制。如下图所示，我们已经成功地在我们的实验室中使用这个系统来分析亲和抗死亡的球员。这些措施包括植物抗凋亡的Bcl - 2的相关athanoGene（袋）的家庭，以及，如BAX已知的哺乳动物的细胞死亡诱导，。此外，我们已经用这个系统来分析具体截断内蛋白质死亡的功能，可提供线索，这些蛋白质可能的翻译后修饰/激活。在这里，我们提出一种快速，敏感的植物为基础的方法，如在初步调查的特定基因的死亡功能第一步。

Watch this Scientific Journal Video about A ÃŽÂ²-glucuronidase GUS Based Cell Death Assay at JoVE.com

A &#946;-glucuronidase GUS Based Cell Death Assay

程序性细胞死亡，DNA梯状或TUNEL法检测，如哺乳动物系统常用的检测往往难以重现植物。 GUS报告制度相结合，我们提出一个快速的，植物的瞬态检测，分析特定基因的潜在死亡属性。

一个β-葡萄糖醛酸酶（GUS）的基细胞死亡含量

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

a-glucuronidase-gus-based-cell-death-assay

Research

JoVE Journal

Biology

A &#946;-glucuronidase (GUS) Based Cell Death Assay

28.0K 次观看.  Texas A&M University. 程序性细胞死亡，DNA梯状或TUNEL法检测，如哺乳动物系统常用的检测往往难以重现植物。 GUS报告制度相结合，我们提出一个快速的，植物的瞬态检测，分析特定基因的潜在死亡属性。

视频: A β-glucuronidase GUS Based Cell Death Assay

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

研究与基于荧光素酶的记者基因检测膜生源

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

科研

生物学

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

双分子荧光互补（附设）检测蛋白质相互作用的洋葱细胞使用的Helios基因枪

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment10. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios &lt;0.5) showing the highest occurrence10. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (&lt; 5% events with cytoplasmic PI staining).

An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.

改性膜联蛋白V /碘化丙啶检测细胞凋亡细胞死亡的准确评估

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

基因小鼠胚胎干细胞的基础含量为心源性诱导效率量化

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

使用早期的小鼠胚胎组织特异性基因的染色质免疫沉淀检测

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

使用LysoTracker检测及鉴别胚胎干细胞在胚胎细胞程序性死亡

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

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 ...

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ultimately kill. Such A/B toxins generally consist of an enzymatically active Asubunit (e.g., ricin toxin A (RTA)) and one or more cell binding Bsubunit(s), which are responsible for toxin binding to specific cell surface receptors. Our current knowledge of how A/B toxins are capable of efficiently intoxicating cells helped scientists to understand fundamental cellular mechanisms, like endocytosis and intracellular protein sorting in higher eukaryotic cells. From a medical point of view, it is likewise important to identify the major toxin trafficking routes to find adequate treatment solutions for patients or to eventually develop therapeutic toxin-based applications for cancer therapy.
Since genome-wide analyses of A/B toxin trafficking in mammalian cells is complex, time-consuming, and expensive, several studies on A/B toxin transport have been performed in the yeast model organism Saccharomyces cerevisiae. Despite being less complex, fundamental cellular processes in yeast and higher eukaryotic cells are similar and very often results obtained in yeast can be transferred to the mammalian situation.
Here, we describe a fast and easy to use reporter assay to analyze the intracellular trafficking of RTA in yeast. An essential advantage of the new assay is the opportunity to investigate not only RTA retro-translocation from the endoplasmic reticulum (ER) into the cytosol, but rather endocytosis and retrograde toxin transport from the plasma membrane into the ER. The assay makes use of a reporter plasmid that allows indirect measurement of RTA toxicity through fluorescence emission of the green fluorescent protein (GFP) after in vivo translation. Since RTA efficiently prevents the initiation of protein biosynthesis by 28S rRNA depurination, this assay allows the identification of host cell proteins involved in intracellular RTA transport through the detection of changes in fluorescence emission.

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

In the manuscript, we describe the use of a yeast-based fluorescence reporter assay to identify cellular components involved in the trafficking and killing processes of the cytotoxic A subunit of the plant toxin ricin (RTA).

一个简单的荧光记者的检测方法, 用于识别蓖麻毒素所需的细胞成分 a 链 (贸易协定) 贩运酵母

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...

The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in the biogenesis of melanosomes, a lysosome-related organelle produced by melanocytes. REs deliver the melanocyte-specific cargo to premature melanosomes during their formation. Blockage in the generation of REs, observed in several mutants of Hermansky-Pudlak syndrome, results in hypopigmentation of skin, hair, and eye. Therefore, studying the dynamics (refer to number and length) of REs is useful to understand the function of these organelles in normal and disease conditions. In this study, we aim to measure the RE dynamics using a resident SNARE STX13.

Recycling endosomes are part of the endosomal tubular network. Here we present a method to quantify the dynamics of recycling endosomes using GFP-STX13 as an organelle marker.

基于显微镜的测定，用于使用SNARE运输研究和分析回收内体

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in ...

A Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. 
In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to &gt;10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in &gt;1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6.

使用基于比色细胞的测定法检测针对 AAV 的中和抗体的分步方法

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health ...