Name: using ustilago maydis as a trojan horse for in situ delivery of maize proteins
Uploaded: 2019-02-08T13:00:00
Description: Watch this Scientific Journal Video about Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins at JoVE.com

作者要感谢托马斯·德雷塞尔豪斯、马丁·帕尼斯克、努雷丁·杰拉和阿明·希尔德布兰德提供的实验室空间和植物材料。关于特洛伊木马方法的最初工作得到了 leopoldina 博士后研究金和 nsf 项目 ios13-9229 的支持。本文介绍的工作得到了 dfg sfb924 (项目 a14 和 b14) 的支持。

1. 建造一个 u. maydis特洛伊木马
请注意:请参见图 1。
<ol><li>利用基因特异性引物和 dna 聚合酶校对, 放大玉米 cdna 中感兴趣的基因。克隆主要 pcr 产物, 并按照质粒供应商的指示将结构转化为大肠杆菌.在使用下一个克隆步骤之前, 通过 sanger 测序验证感兴趣序列的正确基因。 请注意:由于引物序列特性和最佳?...

<ol>
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N., Ziemann, S., Treitschke, S., Assmann, D., Doehlemann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compatibility+in+the+Ustilago+maydis-maize+interaction+requires+inhibition+of+host+cysteine+proteases+by+the+fungal+effector+Pit2.">Compatibility in the Ustilago maydis-maize interaction requires inhibition of host cysteine proteases by the fungal effector Pit2.</a> PLOS Pathogens. 9, e1003177 (2013).</li><li>Ziemann, 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=An+apoplastic+peptide+activates+salicylic+acid+signalling+in+maize.">An apoplastic peptide activates salicylic acid signalling in maize.</a> Nature Plants. 4, 172-180 (2018).</li><li>Ju&#225;rez-Montiel, M., 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+corn+smut+('Huitlacoche')+as+a+new+platform+for+oral+vaccines.">The corn smut ('Huitlacoche') as a new platform for oral vaccines.</a> PLoS One. 10, e0133535 (2015).</li><li>Sarkari, P., Feldbr&#252;gge, M., Schipper, K., Schmoll, M., Dattenb&#246;ck, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Gene Expression Systems in Fungi: Advancements and Applications. , 183-200 (2016).</li><li>Monreal-Escalante, 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+corn+smut-made+cholera+oral+vaccine+is+thermostable+and+induces+long-lasting+immunity+in+mouse.">The corn smut-made cholera oral vaccine is thermostable and induces long-lasting immunity in mouse.</a> Journal of Biotechnology. 234, 1-6 (2016).</li><li>Stock, 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=Applying+unconventional+secretion+of+the+endochitinase+Cts1+to+export+heterologous+proteins+in+Ustilago+maydis.">Applying unconventional secretion of the endochitinase Cts1 to export heterologous proteins in Ustilago maydis.</a> Journal of Biotechnology. 161, 80-91 (2012).</li><li>van der Linde, K., 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=Pathogen+Trojan+horse+delivers+bioactive+host+protein+to+alter+maize+(Zea+mays)+anther+cell+behavior+in+situ.">Pathogen Trojan horse delivers bioactive host protein to alter maize (Zea mays) anther cell behavior in situ.</a> The Plant Cell. 30, 528-542 (2018).</li><li>B&#246;sch, K., 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=Genetic+manipulation+of+the+plant+pathogen+Ustilago+maydis+to+study+fungal+biology+and+plant+microbe+interactions.">Genetic manipulation of the plant pathogen Ustilago maydis to study fungal biology and plant microbe interactions.</a> Journal of Visualized Experiments. , e54522 (2016).</li><li>Chavan, S., Smith, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+efficient+method+for+assessing+pathogenicity+of+Ustilago+maydis+on+maize+and+teosinte+lines.">A rapid and efficient method for assessing pathogenicity of Ustilago maydis on maize and teosinte lines.</a> Journal of Visualized Experiments. 50712, (2014).</li><li>Kelliher, T., Walbot, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+and+patterning+of+the+five+cell+types+of+the+Zea+mays+anther+locule.">Emergence and patterning of the five cell types of the Zea mays anther locule.</a> Developmental Biology. 350, 32-49 (2011).</li><li>Egger, R. L., Walbot, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+Zea+mays.+tassel+development+and+correlation+with+anther+developmental+stages+as+a+guide+for+experimental+studies.">Quantifying Zea mays. tassel development and correlation with anther developmental stages as a guide for experimental studies.</a> Maydica. 60, M34 (2015).</li><li>Holliday, R., King, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bacteria, Bacteriophages, and Fungi: Volume 1. , 575-595 (1974).</li><li>Doehlemann, G., Reissmann, S., A&#223;mann, D., Fleckenstein, M., Kahmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+linked+genes+encoding+a+secreted+effector+and+a+membrane+protein+are+essential+for+Ustilago+maydis-induced+tumour+formation.">Two linked genes encoding a secreted effector and a membrane protein are essential for Ustilago maydis-induced tumour formation.</a> Molecular Microbiology. 81, 751-766 (2011).</li><li>Banuett, F., Herskowitz, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+a+alleles+of+Ustilago+maydis+are+necessary+for+maintenance+of+filamentous+growth+but+not+for+meiosis.">Different a alleles of Ustilago maydis are necessary for maintenance of filamentous growth but not for meiosis.</a> Proceedings of the National Academy of Sciences. 86, 5878-5882 (1989).</li><li>Bortfeld, M., Auffarth, K., Kahmann, R., Basse, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ustilago+maydis+a2+mating-type+locus+genes+lga2+and+rga2+compromise+pathogenicity+in+the+absence+of+the+mitochondrial+p32+family+protein+Mrb1.">The Ustilago maydis a2 mating-type locus genes lga2 and rga2 compromise pathogenicity in the absence of the mitochondrial p32 family protein Mrb1.</a> The Plant Cell. 16, 2233-2248 (2004).</li></ol>

现代作物研究需要关于基因和蛋白质水平的分子分析的协议。对于玉米等大多数作物品种来说,通过转化实现遗传可及性是不可用的, 也是效率低下和耗时的。此外, 可靠的遗传工具, 如启动子报告系统, 这使得很难研究在不同的组织部位具有高时空分辨率的原位蛋白功能。光蛋白可以通过异源表达和纯化的蛋白质渗入组织来研究。然而, 尽管异源蛋白表达取得了进展, 但有针对性地渗入作物组...

生物养病原体乌斯蒂拉戈梅迪斯是玉米黑麦病1的病原体。它感染玉米的所有空中部位, 导致大肿瘤, 其中含有黑色素化的黑色孢子。在全球范围内,据估计, 美国梅迪斯每年会造成玉米产量损失 2% 左右, 而在墨西哥, 肿瘤被认为是一种美食。植物感染是由一种附着物引起的, 这种酶分泌细胞壁裂解酶, 以穿透玉米表皮细胞的第一层。从一个主要的感染部位,美国的梅迪斯生长在细胞内和细胞间, 每天侵入一到两个细胞层 1,2。成功的感染导致植物肥大, 在感染后5天变成可见的肿瘤 1,3,4。在所有感染阶段, 真菌菌丝侵入植物细胞质膜, 而不与宿主细胞质1,2有任何直接接触。感染菌丝与植物质膜之间紧密的表质空间被认为是宿主病原体的互动位点, 称为生物养能相互作用区。为了克服植物固有的免疫系统,美国将一系列效应蛋白分泌到生物养相互作用区 1。一些效应被植物细胞吸收, 而另一些效应则留在生物养细胞相互作用区 5、6、7、8。一个热塑性效应器是 umpit2, 它与热塑性玉米蛋白酶相互作用, 通过热塑性蛋白酶活性9,10防止 zmprozip 中的信号肽 zmzip1 的释放。
在过去的几十年里,由于人们对生命周期的了解、遗传的可及性和分泌物的异种表达, 美国梅迪斯不仅成为植物-病原体相互作用中真菌遗传学的典范, 而且成为生物技术中的一个宝贵工具。蛋白质11,12,13。常规和非常规蛋白质分泌的信号已被确定, 允许控制翻译后的修饰 14.最近,美国梅迪斯被用作特洛伊木马工具, 在原地研究小的、分泌的玉米蛋白.成功地应用特洛伊木马方法分析了参与花药发育的小的分泌蛋白 zmmac1 的功能。zmmac1 诱导新形成的细胞15的多能细胞的周周分裂和细胞命运指标.用同样的方法揭示了玉米损伤相关肽 zmzip1 的生物学功能。美国的梅迪斯分泌玉米 zmzip1 导致肿瘤形成受损10。因此, 特洛伊木马方法是一种有价值的替代途径, 可用于具有较高时空分辨率的原位蛋白质研究, 既不需要生成稳定的玉米转化线, 也不需要异源组织浸润表达和纯化的蛋白质。特别是, 特洛伊木马策略使任何异源蛋白分泌到玉米植物体内, 并直接比较同一组织内受感染的植物细胞与未感染的植物细胞。
该协议说明了产生一个美国梅迪斯特洛伊木马菌株来研究感兴趣的蛋白质的主要步骤。它还包括关于三种不同玉米组织类型 (成年叶子、花被和耳朵) 感染程序的准确信息, 这是研究时空感染进展和蛋白质功能的先决条件在这些目标组织中没有对玉米基因扩增和显微成像技术作出进一步的规定, 因为这些步骤是针对特定目标和与仪器有关的。因此, 该协议是针对有经验的标准分子生物学技术的用户。

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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:307px;">2 mL syringe&#160;</td>
			<td style="width:119px;">B. Braun</td>
			<td style="width:89px;">4606027V</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">23G x 1 1/4 hypodermic needle</td>
			<td style="width:119px;">B. Braun</td>
			<td>4657640</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bacto Peptone&#160;</td>
			<td style="width:119px;">BD</td>
			<td>211677</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">cDNA from maize</td>
			<td style="width:119px;"></td>
			<td style="width:89px;"></td>
			<td style="width:311px;">from maize tissue expressing the gene of interrest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Charcoal</td>
			<td>Sigma-Aldrich</td>
			<td style="width:89px;">05105</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal laser scanning microscope</td>
			<td style="width:119px;"></td>
			<td style="width:89px;"></td>
			<td style="width:311px;">use locally available equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Cuvette (10 x 4 x 45 mm)</td>
			<td style="width:119px;">Sarstedt</td>
			<td style="width:89px;">67742</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator-shaker set to 28 &#176;C, 200 rpm</td>
			<td style="width:119px;"></td>
			<td style="width:89px;"></td>
			<td style="width:311px;">use locally available equipment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">Light microscope with 400-fold magnification</td>
			<td style="width:119px;"></td>
			<td style="width:89px;"></td>
			<td style="width:311px;">use locally available equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Nco I</td>
			<td style="width:119px;">NEB</td>
			<td style="width:89px;">R0193</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="49">
			<td height="49" style="height:49px;">p123-PUmpit2-SpUmpit2-Zmmac1-mCherry-Ha</td>
			<td style="width:119px;"></td>
			<td style="width:89px;"></td>
			<td style="width:311px;">please contact the corresponding author&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Pasteur pipet (glass, long tip)</td>
			<td style="width:119px;">VWR</td>
			<td>14673-043</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">pCR-Blunt-II-TOPO</td>
			<td style="width:119px;">Thermo Fisher Scientific</td>
			<td>K280002</td>
			<td style="width:311px;">can be exchanged for other basic cloning vectors like pENTR or pJET</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potato Dextrose Agar&#160;</td>
			<td style="width:119px;">VWR</td>
			<td style="width:89px;">90000-745</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:307px;">Sharpie pen</td>
			<td style="width:119px;"></td>
			<td style="width:89px;"></td>
			<td style="width:311px;">use locally available equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Spectrophotometer</td>
			<td style="width:119px;"></td>
			<td style="width:89px;"></td>
			<td style="width:311px;">use locally available equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Ssp I</td>
			<td style="width:119px;">NEB</td>
			<td style="width:89px;">R0132</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td style="width:89px;">S0389</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">T4 DNA ligase</td>
			<td style="width:119px;">NEB</td>
			<td style="width:89px;">M0202</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">TRIS</td>
			<td>Sigma-Aldrich</td>
			<td style="width:89px;">TRIS-RO</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Xba I</td>
			<td style="width:119px;">NEB</td>
			<td style="width:89px;">R0145</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast extract&#160;</td>
			<td style="width:119px;">BD</td>
			<td>212750</td>
			<td style="width:311px;"></td>
		</tr>
	</tbody>
</table>

using ustilago maydis as a trojan horse for in situ delivery of maize proteins

在荷马特洛伊木马神话的启发下, 我们设计了玉米病原体乌斯蒂拉戈·梅迪斯,将分泌的蛋白质传递到玉米注入中, 允许在体内进行表型分析。这种方法不依赖于玉米的转化, 而是利用了微生物遗传学和病原体的分泌能力。在这里, 它允许检查体内提供的分泌的蛋白质与高时空分辨率在不同种类的感染部位和组织。特洛伊木马策略可用于瞬时补充玉米功能损失表型, 功能表征蛋白质域, 分析非目标蛋白效应, 或研究单侧蛋白质过量, 使其成为一个强大的工具玉米作物系统中的蛋白质研究。这项工作包含了一个精确的协议, 如何生成特洛伊木马菌株, 然后是标准化的感染协议, 将这种方法应用于三种不同的玉米组织类型。

构建 u. maydis特洛伊木马实验被克隆到质粒 p123-p umpit2-spum pit2-基因的兴趣 mmcherrie-ha.感兴趣的玉米基因与mcherry荧光记者和表位 ha标记融合在一起。融合蛋白的表达在美国的 fydis umpit2启动子的控制下, 该启动子在感染 21期间被特别激活。为了将感兴趣的肽蛋...

Watch this Scientific Journal Video about Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins at JoVE.com

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

使用乌斯蒂拉戈梅迪斯作为特洛伊木马在现场交付玉米蛋白

这项工作描述了克隆一个 ust原 maydis特洛伊木马菌株, 以便在原地传递分泌的玉米蛋白到三种不同类型的玉米组织。

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast ...

研究基因和蛋白质水平的过程在作物研究中至关重要。我们的方法能够检查分泌的迷宫蛋白，在不同的感染边和组织具有最高的空间时间分辨率。特洛伊木马方法探索有用的自动分泌功能。它绕过耗时和具有挑战性的迷宫变换，并促进对迷宫中分泌蛋白质的详细研究。有关添加的迷宫叶、流苏和耳朵正确感染的详细资料。并展示了目标组织中感染进展和蛋白质功能的成功研究。演示这个程序的将是伊莎贝尔·菲德勒，一个来自实验室的研究生。要开始实验，使用无菌巴氏菌移液器从尿板中刮出 Eumadous。接种5毫升的YES光介质与尤马杜斯。在 28 摄氏度下生长培养，在 200 rpm 下持续摇晃 16 小时。第二天，将 900 微升的新鲜 YEPS 灯与 100 微升的隔夜文化混合在一起。在分光光度计分析中，使用 YEPS 光介质作为空白测量 OD-600。用新鲜的 YEPS 光介质稀释过夜文化，使 O-600 的 2。让培养在 28 摄氏度时生长，在 200 rpm 时持续摇晃，直到达到中日志生长阶段，8 到 1.0 的 OD-600 表示。孵育后，通过旋转细胞3000次气10分钟来收获细胞。丢弃上一杯。接下来，用DDH2O的1个培养量洗涤细胞堆。旋转细胞在3000时间气10分钟，并丢弃上一液。然后，使用 20 毫升玻璃移液器在 DDH2O 中小心地重新悬挂细胞桩。因此，调整特洛伊木马检测的最终 OD-600 到 3.0 或 1.0 用于疾病评级。培养迷宫植物到成年叶的阶段，其中至少叶7生长在茎内。接下来，将 Eumads 培养素转移到 3 毫升注射器中，并用 20 量表双皮下针。小心按茎以本地化镜茎组织。镜茎的基座可以通过从较硬的茎向较软的组织的过渡来区分。用钢笔在茎上标记镜茎。注射1.5毫升的尤马杜斯培养1厘米以上的拍摄镜茎或花序镜茎。一旦迷宫植物到达流苏阶段，请小心按茎，将流苏本地化在茎中。用笔标记茎上的流苏尖端和底座。将 Eumads 培养器转移到 3 毫升注射器中，用 20 量双皮下针注射流苏周围总共 1.5 毫升的注射剂。为保证幼崽的均匀分布，请缓慢地将 5 毫升放在笔尖、中间和用笔标记的流苏底座上。将 Eumads 培养器与 20 量双皮下针转移到 3 毫升注射器中。将接种针注射到壳叶之间的空间中，在不伤害耳朵的情况下，释放耳朵周围1.5毫升的接种剂。接下来，取出注射器和针头，并仔细按摩球状，以平均分配 Eumadous 溶液。在 PD 木炭螺旋板上滴 10 微升的 inolum。然后在室温下孵育2天。分泌 ZM Mach1 的 Hyphae 被荧光和樱桃信号包围着。相比之下，非秘密催眠只显示真菌细胞质中的荧光信号。与均匀分布相比，流苏定位不当或配金不均匀会导致未感染流苏或仅部分感染流苏。独奏致病特洛伊木马Progenester应变SG-200，培养在PD木炭螺旋钻，形成白色绒毛丝在板上，如图所示。而尤马杜斯菌株FB1要求在传染性丝形成前交配。感染后，可以使用各种方法（包括疾病评分或显微成像）分析特洛伊木马肝蛋白的可能反应。对于许多病原体，转化和分泌是被充分理解的，可以以类似的方式探索。因此，它们支持对主机的研究，这些主机不易受转换的影响。

Research

JoVE Journal

Genetics

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

植物病原体的遗传操作<em&gt;玉米黑粉菌</em&gt;研究真菌生物学和植物微生物相互作用

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

科研

遗传学

Detection of Copy Number Alterations Using Single Cell Sequencing

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

目标<em&gt;原位</em&gt;组蛋白突变的基因在芽殖酵母

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

高分辨率荧光原位杂交在果蝇胚胎和组织中使用 Tyramide 信号放大

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

使用斑马鱼作为体内模型系统分析 DNA 复制定时

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Canalostomy 为成人和新生小鼠内耳局部药物传递的外科治疗方法

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

全挂载原位杂交法可视化蜱内脏微生物群

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

RNA原位杂交法定量分析小鼠脑切片中的替代前 mRNA 拼接方法

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

原位杂交法在小鼠心脏切片组织特异 miRNA 表达谱分析中的应用

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis

原位杂交的色原性杂交是HPV相关头颈部癌症诊断的工具

Human papillomavirus (HPV) infection is a major risk factor for a subtype of oropharyngeal squamous cell carcinoma (OPSCC), which tends to be associated ...