Name: visualizing and tracking endogenous mrnas in live drosophila melanogaster egg chambers
Uploaded: 2019-06-04T12:30:00
Description: Watch this Scientific Journal Video about Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers at JoVE.com

我们感谢Salvatore A.E.Marras(罗格斯大学公共卫生研究所中心)对分子信标的合成、标记和纯化,并感谢丹尼尔·圣约翰斯顿(剑桥大学古登研究所)的奥斯卡-MS2/ MCP-GFP转基因苍蝇库存。这项工作得到了国家科学基金会CAREER奖1149738和专业员工大会-CUNY奖的支持。

1. 活细胞成像MB的设计
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折叠目标RNA序列,使用mfold服务器(http://unafold.rna.albany.edu/?q=mfold/RNA-Folding-Form)中的"RNA形式"预测mRNA目标的二次结构。
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<li>以 FASTA 格式粘贴/上传目标序列,选择 5 或 10% 次优极性(自由能量在 MFE 值的 5 或 10% 以内折叠的结构),并相应地调整计算折叠的最大数量(例如,较大的 10%次优度)。 注意:在设计MB时,包括次优二级结构,可以识别目标mRNA内可能比?...

<ol>
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果蝇卵室内源性mRNA贩运的实时可视化依赖于使用特定、高效且耐核酸酶的MB,现在可以使用PinMol软件轻松设计。MB 是特定探针,旨在检测目标 mRNA 内的独特序列(最好是无二次结构的区域),从而能够对转录记录进行高解析检测。在其他组织/细胞类型采用此技术/协议时,唯一的限制是感兴趣的样本的 MB 交付效率。虽然其他方法要求对组织进行基因操作,以表达贴合剂和RNA结合蛋白,并带有荧?...

通过开发基于荧光的活细胞成像技术,能够用空间和时间分辨率来可视化动态事件。目前,在体内mRNA可视化是通过基于RNAaptamer-蛋白相互作用、RNAaptamer诱导的有机染料荧光和核酸探退火1、2、3.均提供高特异性、灵敏度和信背景比。然而,以RNA贴合剂为中心的方法需要大量的基因操作,其中转基因被设计为表达具有蛋白质或有机染料结合所需的人工结构图案的RNA。例如,MS2/MCP 系统需要共同表达一个转基因表达的RNA结构,该结构包含噬菌体 MS2 涂层蛋白 (MCP) 的多重串联重复,另一个转基因编码荧光蛋白融合到MCP4,5。在RNA中加入这种二次结构图案,以及大量荧光标记的蛋白质,引起了人们的担忧,即原生RNA过程可能受到影响6。一项解决了这一问题并提供额外独特优势的技术是核酸方法,分子信标(MB)。MB允许多向地源性mRNA检测,区分单核苷酸变异,以及快速动力学与目标mRNA7,8杂交。MB是寡核苷酸探针,一旦杂交到目标(图1C)9,在经历氟性形成变化之前,它们仍停留在淬火发夹褶皱中。几个组已经成功地使用MB检测非编码RNA(微RNA和lncRNA)10,11,12,13,RNA逆转录病毒14和动态DNA蛋白交互15.它们已成功应用于各种生物体和组织成像,如斑马鱼胚胎16、神经元13、肿瘤组织17、区分心肌细胞18和沙门氏菌19.
在这里,我们描述了活D.melanogaster卵室内源性mRNA的设计、交付和检测方法,以及足够快的显微镜设置,能够捕获活性分子传输的动态范围。D. melanogaster卵子室是一个理想的多细胞模型系统,用于广泛的发育研究,从早期生殖系干细胞分裂和母体基因表达到分段体计划20的生成, 21.蛋室易于隔离,大而半透明,能够承受数小时的体外分析,使其高度易于成像实验。在积极翻译之前,许多工作都集中在母体抄本到离散亚细胞区域的不对称本地化上。特别是,oskar mRNA定位及其随后在卵母细胞后极的翻译必须以严格调节的方式发生,以避免致命的双体胚胎表型22。oskar mRNA被转录在15个生殖系细胞中,称为护士细胞,并通过细胞质桥(称为环管)积极输送到卵母细胞,即成为成熟卵子并最终受精的生殖细胞(图1A)).现有大量关于蛋白质因子动态招募和交换的信息,以及其远程细胞内旅行,使oskar成为学习的首选mRNA生命周期的许多过程。MB有助于揭示mRNA定位过程的细节,并破译控制果蝇细胞生成过程中控制mRNA传输的蛋白质因子的调控和功能。特别是,通过将MB微注射到护士细胞中并进行活细胞成像实验,可以追踪内源性mRNA8,23。
此处提供的路线图提供了完整流程的步骤,从使用 MB 进行活细胞成像实验、获取成像数据,到执行数据分析以跟踪其原生细胞环境中的内源性 mRNA。可以修改和进一步优化这些步骤,以满足在他们自己的实验室环境中处理其他组织/细胞类型的研究人员的需求。

<table>
	<tbody>
		<tr>
			<td>Spectrofluorometer</td>
			<td>Fluoromax-4 Horiba-Jobin Yvon</td>
			<td>n/a</td>
			<td>Photon counting spectrofluorometer</td>
		</tr>
		<tr>
			<td>Quartz cuvette</td>
			<td>Fireflysci (former Precision Cells Inc.)</td>
			<td>701MFL</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 tweezer</td>
			<td>World Precision Instruments</td>
			<td>501985</td>
			<td>Thin tweezers are very important to separate out the individual egg chambers</td>
		</tr>
		<tr>
			<td>Halocarbon oil 700</td>
			<td>Sigma-Aldrich</td>
			<td>H8898</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slip No.1 22 x 40mm</td>
			<td>VWR</td>
			<td>48393-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica MZ6 Leica Microsystems Inc.</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 fruit fly anesthesia pad</td>
			<td>Genesee Scienific</td>
			<td>59-114</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCL pH7.5</td>
			<td>Sigma-Aldrich</td>
			<td>1185-53-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>7791-18-6</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinning disc confocal microscope</td>
			<td>Leica DMI-4000B inverted microscope equipped with Yokogawa CSU 10 spinning disc Leica Microsystems Inc.</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamamatsu C9100-13 ImagEM EMCCD camera</td>
			<td>Hamamatsu</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>PatchMan NP 2 Micromanipulator</td>
			<td>Eppendorf Inc.</td>
			<td>920000037</td>
			<td></td>
		</tr>
		<tr>
			<td>FemtoJet Microinjector</td>
			<td>Eppendorf Inc.</td>
			<td>920010504</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection needle: Femtotips II</td>
			<td>Eppendorf Inc.</td>
			<td>930000043</td>
			<td></td>
		</tr>
		<tr>
			<td>Loading tip: 20ul Microloader</td>
			<td>Eppendorf Inc.</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Cover glasses no. 1 or 1.5, 22x40mm</td>
			<td>VWR</td>
			<td>48393-026; 48393-172</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry yeast</td>
			<td>Any grocery store</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Computer, &#62; 20 GB RAM</td>
			<td></td>
			<td></td>
			<td>Although processing can be carried out on most computers, higher capabilities will increase the speed of the processing</td>
		</tr>
	</tbody>
</table>

visualizing and tracking endogenous mrnas in live drosophila melanogaster egg chambers

基于荧光的成像技术,结合光显微镜的发展,彻底改变了细胞生物学家进行活细胞成像研究的方式。自从开创性研究将位点特异性mRNA定位与基因表达调控联系起来以来,检测RNA的方法已经大大扩展。动态 mRNA 过程现在可以通过检测 mRNA 的方法进行可视化,再加上足够快的显微镜设置,可以捕获分子行为的动态范围。分子信标技术是一种基于杂交的方法,能够直接检测活细胞中的内源性转录。分子信标是发夹状、内部淬火、单核苷酸鉴别核酸探针,只有在杂交到独特的靶序列时才会产生荧光。当与先进的荧光显微镜和高分辨率成像相结合时,它们使人们能够对mRNA的细胞内运动进行空间和时间跟踪。虽然这项技术是能够检测内源转录本的唯一方法,但由于在设计活细胞成像的此类探针方面有困难,细胞生物学家尚未完全接受这项技术。一种新的软件应用程序PinMol,允许增强和快速设计的探头最适合有效地杂交到活细胞内的mRNA目标区域。此外,高分辨率、实时图像采集和当前开源图像分析软件可实现精细的数据输出,从而更精细地评估 mRNA 生命周期中涉及的动态过程的复杂性。
在这里,我们提出了一个全面的协议,用于设计和交付分子信标到果蝇黑色素细胞的卵子室。通过旋转盘共聚焦显微镜对内源性母体mRNA进行直接和高度特异性的检测和可视化。成像数据使用冰面软件中的物体检测和跟踪进行处理和分析,以获取有关 mRNA 动态移动的详细信息,mRNA 被传输并本地化到卵母细胞内的专门区域。

使用PinMol,可以为一个 mRNA 目标设计多个 MB(图 1B-C)。经过合成和纯化,使用体外分析对选定的MB进行特征描述和比较。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58545/58545fig1.jpg"> 图1:内源性mRNA活细胞成像技术和组织描述。(A) 用于显微注射的中期果蝇?...

Watch this Scientific Journal Video about Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers at JoVE.com

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

在这里,我们提出了一个协议,用于可视化,检测,分析和跟踪内源性mRNA贩运在活的果蝇黑色素细胞卵室使用分子信标,旋转盘共聚焦显微镜,和开源分析软件。

可视化和跟踪活果蝇黑色素细胞卵室的内源性 mRNA

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...

该方法有助于回答RNA生物学领域有关极化RNA定位机制和功能的关键问题。该技术的主要优点是，它允许实时可视化活组织内源性RNA贩运。该技术的影响延伸到RNA相关疾病的治疗，因为它有可能发现新的治疗靶点。虽然这种方法可以提供对果蝇蛋室RNA过程的洞察，但它也可以应用于其他系统，如斑马鱼或培养细胞。一般来说，由于分子信标道具的设计和交付带来的挑战，新方法的个体将难以克服。对于分子信标微注射，选择 40 倍油目标，并在显微镜舞台上安装带解剖蛋室的盖玻片。找到一个卵室在中后期发育阶段，是适当的定向微注射。然后设置具有适当注射和补偿压力的微注射器。使用微型操纵器操纵手柄，轻轻将装有一微升分子信标溶液的针头放入油滴中。将尖端聚焦到视场的外围。执行清洁功能，从针尖上去除空气，并确保针头流出，并将针头带到家中位置。聚焦蛋室进行微注射，使针头回到蛋室边缘附近的焦点。对目标的 z 位置进行微调，使分离毛囊细胞与护士细胞的膜处于焦点位置。将针头插入护士细胞，在两到五秒内注射分子信标。为确保可重复的微注射成功，请仔细关注分离毛囊细胞和将注射的护士细胞的膜，同时将针头尖与微操纵器聚焦。当所有分子信标都注射后，轻轻取出针头并将其缩回家中位置。将目标更改为所需的放大倍率以进行图像采集。然后专注于新目标的蛋室，开始图像采集。要对分子信标进行现场检测，请打开图像 J 中的图像，使用转换为 ICY 工具将图像转换回 ICY。比例杆将自动覆盖到堆栈上。必要时通过检查器窗口编辑比例线。然后停用图层选项卡下比例栏的眼部图标，从原始堆栈中删除比例条。选择检测和跟踪、检测和现场检测器，并分别为输入和预处理参数选择电流序列输入检测和通道零。对于探测器，选择检测暗背景上的亮点，使用强制使用 2D 波长进行 3D 时，只有在堆栈中没有足够的 z 切片来执行分析时。为每个比例选择比例和灵敏度，为较大的点添加更多比例，并确认序列中感兴趣的区域已设置为感兴趣区域。对于筛选，请确认未设置筛选，并在输出下选择适当的文件格式。如果要将现场检测结果用于跟踪分析，请选择导出到游泳池。对于同化分析，重复其他通道的点检测。要跟踪分子信标点，请选择检测和跟踪、跟踪、点跟踪，并使用与点检测相同的参数运行点探测器。单击估计参数，并在参数估计弹出窗口中选择所需的目标运动。然后单击"运行跟踪"。要可视化轨道，请选择检测和跟踪、跟踪和跟踪管理器。对于颜色轨道处理器，选择启用并选择轨道的颜色。从添加轨道处理器，选择轨道处理器时间剪辑。在检查剪子窗口中，输入当前时间点之前和之后要显示的检测数。将轨道信息另存为 XML 跟踪文件，并使用相机图标获取结果的屏幕截图。要沿 z 方向投影堆栈，请使用搜索栏查找插件，然后从下拉菜单中选择最大投影。在检查器窗口中，选择序列选项卡、画布和旋转以将图像调整到所需方向，并获取旋转图像的屏幕截图。然后选择感兴趣区域、2D 感兴趣区域，选择感兴趣区域形状，并在图像上选择感兴趣区域进行裁剪。安装时间戳叠加插件，并按照弹出窗口中的说明添加时间戳，以了解如何放置和格式化时间戳。若要更改或添加时间间隔，请单击序列属性窗口中的编辑。激活眼睛图标以在检查器窗口中的图层选项卡下添加比例条。然后获取时间戳结果的屏幕截图，并同时以 TIFF 和 AVI 格式保存图像。使用这种方法，如所证明的，可以可视化内源mRNA的传输和定位模式，通过分子信标在不同阶段的卵巢生成，特别是在中生生。当单独注射到同一阶段的蛋室时，不同的奥斯卡特异性分子信标呈现相同的定位模式。注入护士细胞细胞的分子信标将自由扩散到相邻的护士细胞中以及卵巢细胞中。因此，信标能够与目标混合，并在微注射场址以外的其他地点生成荧光信号。在oskar GFP转基因蛋室中，采集数据的分析表明，基因工程GFP标记的oskar mRNA的荧光信号与使用分子信标检测的荧光信号具有广泛的结肠化。此外，5D堆栈可以进一步分析，以确定oskar mRNA轨迹，用于在护士细胞和卵母细胞细胞质的长距离传输。该技术开发后，为RNA生物学领域的研究人员探索果蝇卵室的内源性母体mRNA的运输和定位铺平了道路。

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JoVE Core

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Cell Biology

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Unit 1

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Control of Gene Expression

SCIENTISTS IN ACTION

Dissection of Larval CNS in Drosophila Melanogaster

在果蝇幼虫中枢神经系统的剖析

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

科研

生物学

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

高分辨率视频跟踪移动的，在成年果蝇。

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

Visualizing the Live Drosophila Glial-neuromuscular Junction with Fluorescent Dyes

可视化的Live果蝇胶质细胞神经​​肌肉交界处，与荧光染料

Our project identified GFP labeled glial structures at the developing larval fly neuromuscular synapse. To look at development of live glial-nerve-muscle ...

Visualizing the Beating Heart in Drosophila

Visualizing the Beating Heart in Drosophila

可视化心脏跳动果蝇</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Isolation of Drosophila melanogaster Testes

Isolation of Drosophila melanogaster Testes

分离果蝇睾丸

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Preparing Individual Drosophila Egg Chambers for Live Imaging

准备个人果蝇蛋钱伯斯实时成像

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Measurement of Lifespan in Drosophila melanogaster

Measurement of Lifespan in Drosophila melanogaster

寿命测量<em&gt;果蝇</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Maintenance of a Drosophila melanogaster Population Cage

Maintenance of a Drosophila melanogaster Population Cage

的维持<em&gt;果蝇</em&gt;人口笼

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...