Name: single-cell analysis of the expression of pseudomonas syringae genes within the plant tissue
Uploaded: 2022-10-06T15:00:00
Description: Watch this Scientific Journal Video about Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue at JoVE.com

这项工作得到了由MCIN/AEI/10.13039/501100011033/资助的项目赠款RTI2018-095069-B-I00和“ERDP创造欧洲的方式”的支持。J.S.R.由安达卢兹调查、发展和创新计划（PAIDI 2020）资助。NLP由安达卢兹调查、发展和创新计划的P18-RT-2398项目资助。

1. 植物准备
<ol><li>按照以下步骤准备拟南芥 Col-0 植物。<ol><li>用先前浇水的1：3蛭石 - 植物基质混合物（见 材料表）填充直径10厘米的花盆，并用15厘米x 15厘米的金属网覆盖花盆，并带有1.6毫米x 1.6毫米的孔。使用橡皮筋将金属网调整到潮湿的土壤上（图1A）。</li>
		<li>用湿牙签将拟南芥种子播种到金属网的孔中。将三到四颗种子放在花盆内远处（<...

<ol>
	<li>Weigel, W. A., Dersch, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+heterogeneity:+A+bacterial+virulence+strategy.">Phenotypic heterogeneity: A bacterial virulence strategy.</a> Microbes and Infection. 20 (9-10), 570-577 (2018).</li><li>Hare, P. J., LaGree, T. J., Byrd, B. A., DeMarco, A. M., Mok, W. W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+technologies+to+study+phenotypic+heterogeneity+and+bacterial+persisters.">Single-cell technologies to study phenotypic heterogeneity and bacterial persisters.</a> Microorganisms. 9 (11), 2277 (2021).</li><li>Mansfield, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Top+10+plant+pathogenic+bacteria+in+molecular+plant+pathology.">Top 10 plant pathogenic bacteria in molecular plant pathology.</a> Molecular Plant Pathology. 13 (6), 614-629 (2012).</li><li>Morris, C. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+life+history+of+the+plant+pathogen+Pseudomonas+syringae+is+linked+to+the+water+cycle.">The life history of the plant pathogen Pseudomonas syringae is linked to the water cycle.</a> The ISME Journal. 2 (3), 321-334 (2008).</li><li>Rufi&#225;n, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+microscopy+reveals+in+planta+dynamic+interactions+between+pathogenic,+avirulent+and+non-pathogenic+Pseudomonas+syringae+strains.">Confocal microscopy reveals in planta dynamic interactions between pathogenic, avirulent and non-pathogenic Pseudomonas syringae strains.</a> Molecular Plant Pathology. 19 (3), 537-551 (2018).</li><li>Macho, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subversion+of+plant+cellular+functions+by+bacterial+type-III+effectors:+Beyond+suppression+of+immunity.">Subversion of plant cellular functions by bacterial type-III effectors: Beyond suppression of immunity.</a> New Phytologist. 210 (1), 51-57 (2016).</li><li>Xiao, Y., Hutcheson, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+promoter+sequence+recognized+by+a+newly+identified+alternate+sigma+factor+directs+expression+of+pathogenicity+and+host+range+determinants+in+Pseudomonas+syringae.">A single promoter sequence recognized by a newly identified alternate sigma factor directs expression of pathogenicity and host range determinants in Pseudomonas syringae.</a> Journal of Bacteriology. 176 (10), 3089-3091 (1994).</li><li>Rufi&#225;n, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+chromosome-located+transcriptional+fusions+to+fluorescent+proteins+for+single-cell+gene+expression+analysis+in+Pseudomonas+syringae.">Generating chromosome-located transcriptional fusions to fluorescent proteins for single-cell gene expression analysis in Pseudomonas syringae.</a> Methods in Molecular Biology. 1734, 183-199 (2018).</li><li>Rufian, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+syringae+differentiates+into+phenotypically+distinct+subpopulations+during+colonization+of+a+plant+host.">Pseudomonas syringae differentiates into phenotypically distinct subpopulations during colonization of a plant host.</a> Environmental Microbiology. 18 (10), 3593-3605 (2016).</li><li>Katagiri, F., Thilmony, R., He, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Arabidopsis+thaliana-Pseudomonas+syringae+interaction.">The Arabidopsis thaliana-Pseudomonas syringae interaction.</a> Arabidopsis Book. 1, 0039 (2002).</li><li>Fouts, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomewide+identification+of+Pseudomonas+syringae+pv.+tomato+DC3000+promoters+controlled+by+the+HrpL+alternative+sigma+factor.">Genomewide identification of Pseudomonas syringae pv. tomato DC3000 promoters controlled by the HrpL alternative sigma factor.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (4), 2275-2280 (2002).</li><li>Huber, P. 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> Robust Statistics. , (1981).</li><li>Freed, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+screen+to+identify+promoters+conferring+high+levels+of+phenotypic+noise.">A simple screen to identify promoters conferring high levels of phenotypic noise.</a> PLoS Genetics. 4 (12), 1000307 (2008).</li><li>Hirano, S. S., Upper, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacteria+in+the+leaf+ecosystem+with+emphasis+on+Pseudomonas+syringae-a+pathogen,+ice+nucleus,+and+epiphyte.">Bacteria in the leaf ecosystem with emphasis on Pseudomonas syringae-a pathogen, ice nucleus, and epiphyte.</a> Microbiology and Molecular Biology Reviews. 64 (3), 624-653 (2000).</li><li>Lindeberg, M., Myers, C. R., Collmer, A., Schneider, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roadmap+to+new+virulence+determinants+in+Pseudomonas+syringae:+Insights+from+comparative+genomics+and+genome+organization.">Roadmap to new virulence determinants in Pseudomonas syringae: Insights from comparative genomics and genome organization.</a> Molecular Plant Microbe Interactions. 21 (6), 685-700 (2008).</li><li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+leaf+infiltration+assay+for+fine+characterization+of+plant+defense+responses+using+the+Arabidopsis+thaliana-Pseudomonas+syringae+pathosystem.">Bacterial leaf infiltration assay for fine characterization of plant defense responses using the Arabidopsis thaliana-Pseudomonas syringae pathosystem.</a> Journal of Visualized Experiments. (104), e53364 (2015).</li><li>O'Leary, B. M., Rico, A., McCraw, S., Fones, H. N., Preston, G. 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+infiltration-centrifugation+technique+for+extraction+of+apoplastic+fluid+from+plant+leaves+using+Phaseolus+vulgaris+as+an+example.">The infiltration-centrifugation technique for extraction of apoplastic fluid from plant leaves using Phaseolus vulgaris as an example.</a> Journal of Visualized Experiments. (94), e52113 (2014).</li><li>Tornero, P., Dangl, J. 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+high-throughput+method+for+quantifying+growth+of+phytopathogenic+bacteria+in+Arabidopsis+thaliana.">A high-throughput method for quantifying growth of phytopathogenic bacteria in Arabidopsis thaliana.</a> The Plant Journal. 28 (4), 475-481 (2001).</li></ol>

这里介绍的方法描述了一种非侵入性程序，它允许细菌渗透到植物叶面组织中，允许快速接种大量，同时最大限度地减少组织破坏。 丁香假单胞菌 物种复合体的特征之一是能够在植物质外体内和植物表面作为附生植物14存活和增殖。因此，不能排除使用本协议提取的细菌仅来自植物质外体的可能性。然而，以前使用共聚焦显微镜证明，与植物叶片内的细菌生长相比，叶子...

致病菌在不同表型中表现出差异，导致在基因相同的种群中形成亚群。这种现象被称为表型异质性，并已被提出作为细菌-宿主相互作用期间的适应策略1。共聚焦显微镜、流式细胞术和微流体的光学分辨率与荧光蛋白相结合的最新进展促进了细菌种群的单细胞分析2。
革兰氏阴性丁香假单胞菌是一种原型植物致病菌，由于其学术和经济重要性3。丁香假单胞菌的生命周期与水循环4有关。丁香假单胞菌通过气孔或伤口等自然孔径进入叶肉细胞之间的细胞间空间，即植物叶质外体5。一旦进入质外体，丁香假单胞菌依靠III型分泌系统（T3SS）和III型易位效应器（T3E）来抑制植物免疫并操纵植物细胞功能以造福病原体6。T3SS和T3E的表达取决于主调节因子HrpL，这是一种与靶基因7启动子区域中hrp盒基序结合的替代西格玛因子。
通过产生与目的基因下游荧光蛋白基因的染色体定位转录融合，可以根据单细胞水平8发射的荧光水平监测基因表达。使用这种方法，已经确定 hrpL 的表达在实验室中生长的细菌培养物和从植物质外体8，9中回收的细菌种群中都是异质的。虽然单细胞水平的基因表达分析通常在实验室培养基中生长的细菌培养物中进行，但这种分析也可以对植物内生长的细菌种群进行，从而为自然环境中亚群的形成提供有价值的信息。分析从植物中提取的细菌种群的潜在限制是，经典的接种方法通过注射器压力浸润到质外体中，然后通过浸渍叶组织进行细菌提取，通常会产生大量干扰下游分析的细胞植物碎片10。大多数细胞碎片由叶绿体的自发荧光片段组成，这些片段与GFP荧光重叠，导致误导性结果。 
本协议描述了分析两个模型病理系统中单细胞基因表达异质性的过程：由 丁香假单胞菌 pv形成的病理系统。番茄菌株DC3000和 拟南芥 （Col-0），另一种由 丁香假单胞 菌PV组成。菜豆菌株1448A和豆类植物（菜豆 品种加拿大奇迹）。提出了一种基于真空渗透的接种方法，利用真空室和泵，形成了一种快速、无损的全叶浸润方法。此外，作为对传统方案的改进，使用一种更温和的方法从质外体中提取细菌群，从而显着减少组织破坏，基于通过在注射器内使用少量体积施加正压和负压循环来提取质外体液。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.17 mm coverslip</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>1.6 x 1.6 mm metal mesh</td>
			<td>Buzifu</td>
			<td></td>
			<td>Fiberglass screen mesh</td>
		</tr>
		<tr>
			<td>10 cm diameter pots</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>140 mm Petri dishes</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>20 mL syringe</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Sarstedt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Merk</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin sodium</td>
			<td>GoldBio</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteriological agar</td>
			<td>Roko</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope Stellaris</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACSVerse cell analyzer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin sulfate</td>
			<td>Duchefa</td>
			<td>G-0124</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin monosulfate</td>
			<td>Phytotechnology</td>
			<td>K378</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Merk</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Merk</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Pechiney Plastic Packaging</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plant substrate</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Silwet L-77</td>
			<td>Cromton Europe Ltd</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Toothpicks</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Merk</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Vacuum chamber 25 cm diameter</td>
			<td>Kartell</td>
			<td>554</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>GAST</td>
			<td>DOA-P504-BN</td>
			<td></td>
		</tr>
		<tr>
			<td>Vermiculite</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>Merk</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell analysis of the expression of pseudomonas syringae genes within the plant tissue

大量的病原微生物不断攻击植物。 丁香假单胞 菌物种复合体包括与多种宿主特别相关的革兰氏阴性植物致病菌。 丁香假单胞 菌从叶表面进入植物，并在质外体内迅速繁殖，形成占据细胞间隙的微菌落。细菌对荧光蛋白的组成性表达允许微菌落的可视化和在微观水平上监测感染的发展。单细胞分析的最新进展揭示了克隆同基因细菌群体所达到的巨大复杂性。这种复杂性被称为表型异质性，是细菌群落之间基因表达的细胞间差异（与遗传差异无关）的结果。为了在单细胞水平上分析单个位点的表达，荧光蛋白的转录融合已被广泛使用。在胁迫条件下，例如在植物质外体定植期间发生的条件， 丁香假单胞菌 根据关键毒力基因（即 Hrp III 型分泌系统）的异质表达分化为不同的亚群。然而，由于接种和细菌提取过程中固有的机械破坏过程中释放的细胞碎片，因此对从植物组织回收的任何给定丁 香假单胞菌 种群的单细胞分析具有挑战性。本报告详细介绍了一种在拟南芥和豆类植物定植期间在单细胞水平上监测感兴趣的 丁香假单胞菌 基因表达的方法。描述了植物的制备和用于使用真空室接种的细菌悬浮液。这里还解释了通过质外体液提取从感染叶片中回收内生细菌的方法。细菌接种和细菌提取方法均经过经验优化，以最大限度地减少植物和细菌细胞损伤，从而产生最适合显微镜和流式细胞术分析的细菌制剂。

III型分泌系统的表达对于植物内的细菌生长至关重要。T3SS基因的及时表达是通过复杂的调控实现的，其中心是卵胞浆外功能（ECF）西格玛因子HrpL，它是T3SS相关基因表达的关键激活因子11。先前使用染色体定位转录融合到下游无启动子gfp基因并通过共聚焦显微镜和流式细胞术9跟踪表达模式对hrpL的表达进行分析。这些融合是通过PCR和传统克隆产生的...

Watch this Scientific Journal Video about Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue at JoVE.com

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

本协议描述了一种方法，该方法允许对植物质外体内生长的丁 香假单胞菌 种群进行单细胞基因表达分析。

丁香假单胞菌基因在植物组织中表达的单细胞分析

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic ...

我们的方案提供了一种温和的植物接种和提取方法，可以恢复在叶质体中生长的细菌种群，适用于单细胞基因表达分析，如流式细胞术。该方法允许从质外体中提取大量细菌，而不会用植物细胞碎片显着污染样品。这种优化的质外体细菌回收方法可以直接应用于或对包括植物致病菌或有益内吞菌在内的许多植物细菌系统进行微小修改。演示该程序的将是来自我们实验室的博士生Nieves Lopez-Pagan。首先，用先前浇水的 1：3 蛭石植物基质混合物填充一个 10 厘米直径的花盆。用穿孔金属网盖住锅，并使用橡皮筋将金属网调整到潮湿的土壤上。接下来，用湿牙签将拟南芥种子播种到金属网的孔中。将三到四颗种子放在花盆内远处的位置。用塑料圆顶盖住锅以保持较高的相对湿度，并在 4 摄氏度下孵育分层 72 小时。在短日照条件下将花盆转移到植物生长室中。要揭开锅盖，请取下塑料圆顶。种子发芽后，用镊子去除大部分幼苗，在花盆的每个位置保留一棵幼苗。然后用湿纸巾覆盖培养皿的底部并将豆子种子放在上面来准备菜豆植物。用手术胶带密封培养皿，并在28摄氏度下孵育三到四天。将发芽的种子转移到装有湿的1：3蛭石植物基质混合物的10厘米直径的花盆中，并在长时间设置的植物生长室中孵育。将目标丁香假单胞菌菌株的甘油原液从零下 80 摄氏度划出到补充有适当抗生素的 LB 平板中。将板在 28 摄氏度下孵育 40 至 48 小时。刮擦细菌生物质并将其重悬于五毫升10毫摩尔氯化镁中。测量600纳米处的光密度，并通过添加10毫摩尔氯化镁将其调节至0.1。在 10 毫摩尔氯化镁中进行连续稀释，以获得每毫升 10 至 5 CFU 的最终接种浓度的五倍。然后，为拟南芥植物准备200毫升接种物，为豆类植物准备50毫升接种物。接种前，加入表面活性剂Silwet L-77至豆类接种终浓度为0.02%，拟南芥终浓度为0.01%。对于拟南芥浸润，将两根形成X形的木棍放在锅上，并将锅朝下放在直径14厘米的培养皿上，培养皿中含有200毫升接种物。对于豆叶接种，将植物和接种液插入真空室，并将叶子引入含有接种物的50毫升锥形离心管中。给予 500 毫巴的脉冲 30 秒以渗透叶子。用一张纸排出多余的接种液，然后将计划放回相应的生长室。接种后四天，切下拟南芥植物的地上部分或豆类植物的接种叶，并将其放入20毫升注射器中，无需针头。对于豆叶，将叶子自己卷起来，让远轴面朝外。加入足够体积的蒸馏水以覆盖组织。然后，将固定注射器的柱塞插入直立位置，并通过轻轻敲击注射器直到所有空气都位于尖端附近来去除注射器内部多余的空气和气泡，并在取出空气后，用石蜡膜覆盖注射器筒的尖端。现在小心地按压柱塞以产生正压，直到组织变黑。然后，拉动柱塞以产生负压。取出石蜡膜和柱塞，收集含有质外体提取细菌的液体。对于单细胞分析，在蒸馏水中制备1.5%琼脂糖溶液。融化后，添加足够的体积以填充并排放置的两个显微镜载玻片之间的空间，并将另一张载玻片放在顶部。让他们开车 15 分钟，然后小心地取下放在顶部的滑梯。使用刀片，在使用前将琼脂糖垫切成五毫米乘五毫米的碎片。同时，离心一毫米的质外体提取的细菌。使用移液管小心地除去上清液，并将沉淀重悬于20微升水中以浓缩细胞。将两微升浓缩细胞滴在0.17毫米盖玻片上，并用一块琼脂糖垫覆盖液滴。在共聚焦显微镜下可视化细菌制备，以使用特定激光识别绿色荧光细菌。使用明场识别所有细菌并合并两个场。要通过流式细胞术进行分析，请等分试样提取的质外体提取的细菌悬浮液。使用流式细胞仪进行分析并获取 100， 000 个事件。通过GFP荧光监测hrpL GFP的表达。点阵图显示了GFP与细胞大小在群体中的荧光强度分布，而直方图显示了GFP的荧光强度与细胞计数的关系。hrpL ON百分比高于相应的hrpL OFF百分比。 荧光显微镜图像显示与hrpL表达相关的GFP的异质水平。观察到显示低或没有GFP荧光的细菌。为了获得最佳效果，在细菌提取步骤中要温和，以避免损坏植物组织，从而限制植物细胞含量的污染。获得的细菌样品可用于减少植物污染具有优势的其他目的，例如用于后续细菌基因组或转录组学分析的核酸提取。

Research

JoVE Journal

Biology

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

Single Cell Electroporation in vivo within the Intact Developing Brain

在体内的单细胞电穿孔内完整的大脑发育

Single-cell electroporation (SCE) is a specialized technique allowing the delivery of DNA or other macromolecules into individual cells within intact ...

科研

生物学

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

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 ...

In vitro Reconstitution of the Active T. castaneum Telomerase

In vitro Reconstitution of the Active T. castaneum Telomerase

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 ...

Expression Analysis of Mammalian Linker-histone Subtypes

哺乳动物链接组蛋白亚型表达分析

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

荧光显微镜筛选和新一代测序：参与细胞器完整性的基因鉴定的有用工具

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

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

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 ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

成像使用烟草瞬时表达系统MAPK信号通路的空间重组

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

CUBIC协议可视化在全山皮肤配方单细胞分辨率蛋白表达

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Visualizing Stromule Frequency with Fluorescence Microscopy

可视化Stromule频率与荧光显微镜

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

在出生后的小鼠听泡解剖：中耳骨骼和组织学分析的分离

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...