Name: defining the program of maternal mrna translation during in vitro maturation using a single oocyte reporter assay
Uploaded: 2021-06-16T13:00:00
Description: Watch this Scientific Journal Video about Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay at JoVE.com

这项工作得到了NIH R01 GM097165、GM116926和尤尼斯·肯尼迪·施莱佛·尼赫德国家生殖和不孕症转化研究中心P50 HD055764的支持。恩里科·达尔代洛得到了拉勒基金会的奖学金的支持，纳塔莎·科斯特曼斯得到了荷兰科学研究组织（NWO）的鲁比肯研究金的支持。

涉及动物的实验程序得到了旧金山加州大学动物护理和使用委员会（AN182026号协议）的批准。
1. 媒体准备
<ol><li>添加表 1中描述的所有组件，使基本的卵母细胞收集介质和卵母细胞成熟介质。对于基本的收集介质，将 pH 设置为 7.4。对于收集和成熟介质，在使用当天添加 3 毫克/mL 的牛血清白蛋白 （BSA） 和 1 μM 西洛酰胺。</li>
</ol>2. 为 Ypet-3' UTR ?...

<ol>
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提出的方法描述了一种策略，研究在体外卵母细胞成熟期间不同过渡时对目标mRNA的活化和抑制。IL-7，一种细胞因子释放的卵母细胞，可能涉及卵母细胞-cumulus细胞通信8，13，被选择用于描述这种方法。IL-7 在卵母细胞成熟8期间被越来越多地翻译，并且允许使用此方法对转化激活进行良好的可视化。但是，如果在整个实验中以恒?...

一个完全生长的哺乳动物卵母细胞在准备受精和获得托蒂波特时经历了快速变化。这些变化对于受精后维持胚胎发育至关重要。虽然与核成熟相关的事件描述得相对较好，但关于卵母细胞细胞质中的分子过程和通路的了解却要少得多。在卵母细胞成熟期的最后阶段，卵母细胞是转录沉默的，基因表达完全依赖于mRNA的转化和降解1，2。因此，蛋白质的合成对发育能力至关重要，它依赖于在卵母细胞生长1、3早期合成的长寿mRNA的定时翻译程序。作为确定母体mRNA翻译程序的一部分，在母细胞成熟和卵母细胞到卵母细胞的过渡期间执行，本文提出了一个策略，研究在体外美化成熟期间单卵母mRNA在单卵母细胞中的活化和抑制。
在这种方法中，YPet开读帧被克隆到3'UTR的感兴趣成绩单上游。接下来，mRNA编码本报记者是微注入卵母细胞与多基化mRNA编码mCherry控制注射量。使用延时显微镜 测量体外 卵母细胞成熟期间的记者积累。黄色荧光蛋白 （YFP） 和 mCherry 的积累记录在单个卵母细胞中，YFP 信号通过共同注入的 mCherry 的稳定水平进行校正。数据采集后，通过计算曲线拟合获得的曲线斜率， 计算体外 卵母细胞成熟期间的不同时间间隔的翻译率。
此方法提供了一个工具，以实验性地确认所选内源 mRNA 的翻译更改。此外，该方法还通过操纵目标 mRNA 4、5、6 的 3' UTR 的 cis-监管元素，促进控制卵母细胞成熟期间翻译的监管元素的定性。操纵多（A）尾长度也允许洞察腺酶/死细胞5的活动。cis-代理元素或RNA免疫沉淀的突变可用于研究与同位素RNA结合蛋白6，7的相互作用。此外，该方法还可用于通过测量与卵母细胞质量下降8、9、10相关的模型中的目标 3' UTR 翻译来识别对卵母细胞发育能力至关重要的翻译程序的基本组件。本方法论文提出了一个具有代表性的实验，其中21天大的C57/BL6小鼠的卵母细胞被微注射与IL-7的3'UTR融合的Ypet记者。介绍了卵母细胞注射、延时记录和数据分析的设置和协议。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Preparation of media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin Powder Bioxtra</td>
			<td>Sigma-Aldrich</td>
			<td>SIAL-A3311</td>
			<td></td>
		</tr>
		<tr>
			<td>Cilostamide</td>
			<td>EMD Millipore</td>
			<td>231085</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM alpha</td>
			<td>Gibco</td>
			<td>12561-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium Eagle&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>M2645</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin 100x Solution, Sterile Filtered</td>
			<td>Genesee Scientific Corporation (GenClone)</td>
			<td>25-512</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate&#160;</td>
			<td>JT-Baker</td>
			<td>3506-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Gibco&#160;</td>
			<td>11360-070</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure distilled water</td>
			<td>Invitrogen</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Preparation of mRNA encoding YFP/3&#39; UTR and mCherry</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Apex Biomedical&#160;</td>
			<td>20-102QD</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbenicillin disodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>C1389-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Choo-Choo Cloning Kit</td>
			<td>McLab</td>
			<td>CCK-20</td>
			<td></td>
		</tr>
		<tr>
			<td>CutSmart Buffer (10x)</td>
			<td>New England Biolabs</td>
			<td>B7204</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA loading dye (6x)</td>
			<td>Thermo Scientific</td>
			<td>R0611</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP Solution</td>
			<td>New England Biolabs</td>
			<td>N0447S</td>
			<td></td>
		</tr>
		<tr>
			<td>DpnI</td>
			<td>New England Biolabs</td>
			<td>R0176</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneRuler 1 kb DNA ladder</td>
			<td>Thermo Fisher</td>
			<td>SM1333</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar Plates with 100 &#181;g/mL Carbenicillin, Teknova&#160;</td>
			<td>Teknova</td>
			<td>L1010</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Medium (Capsules)</td>
			<td>MP Biomedicals</td>
			<td>3002-021</td>
			<td></td>
		</tr>
		<tr>
			<td>MEGAclear Transcription Clean-Up Kit</td>
			<td>Life Technologies</td>
			<td>AM1908</td>
			<td></td>
		</tr>
		<tr>
			<td>MfeI-HF restriction enzyme</td>
			<td>New England Biolabs</td>
			<td>R3589</td>
			<td></td>
		</tr>
		<tr>
			<td>mMESSAGE mMACHINE T7 Transcription Kit</td>
			<td>Invitrogen</td>
			<td>AM1344</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High Fidelity DNA polymerase</td>
			<td>New England Biolabs</td>
			<td>M0530</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(A) Tailing kit</td>
			<td>Invitrogen</td>
			<td>AM1350</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit&#160;</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td></td>
		</tr>
		<tr>
			<td>S.O.C. medium</td>
			<td>Thermo Fisher</td>
			<td>15544034</td>
			<td></td>
		</tr>
		<tr>
			<td>TAE buffer&#160;</td>
			<td>Apex Biomedical&#160;</td>
			<td>20-193</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure Ethidium Bromide Solution</td>
			<td>Life Technologies</td>
			<td>15585011</td>
			<td></td>
		</tr>
		<tr>
			<td>Oocyte collection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirator tube assembly for calibrated micro-pipettes</td>
			<td>Sigma-Aldrich</td>
			<td>A5177-5EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Calibrated micro-pipettes</td>
			<td>Drummond Scientific Company</td>
			<td>2-000-025&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSG- 5000</td>
			<td>Mybiosource</td>
			<td>MBS142665</td>
			<td></td>
		</tr>
		<tr>
			<td>PrecisionGlide Needle 26 G x 1/2</td>
			<td>BD</td>
			<td>305111</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 1 ml</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>Oocyte micro-injection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Dish | No. 0 Coverslip | 20 mm Glass Diameter | Uncoated</td>
			<td>MatTek</td>
			<td>P35G-0-20-C</td>
			<td>For time-lapse microscopy</td>
		</tr>
		<tr>
			<td>Borosilicate glass with filament</td>
			<td>Sutter Instrument</td>
			<td>BF100-78-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Oil for Embryo Culture</td>
			<td>Irvine Scientific</td>
			<td>9305</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish</td>
			<td>Falcon</td>
			<td>351006</td>
			<td>For micro-injection</td>
		</tr>
		<tr>
			<td>Tissue Culture Dish</td>
			<td>Falcon</td>
			<td>353001</td>
			<td>For oocyte incubation</td>
		</tr>
		<tr>
			<td>VacuTip Holding Capillary</td>
			<td>Eppendorf</td>
			<td>5195000036</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biorender</td>
			<td>BioRender</td>
			<td></td>
			<td>Preparation of Figure 1S</td>
		</tr>
		<tr>
			<td>MetaMorph, version 7.8.13.0&#160;</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>&#160;For time-lapse microscopy, analysis of 3&#39; UTR translation&#160;</td>
		</tr>
	</tbody>
</table>

defining the program of maternal mrna translation during in vitro maturation using a single oocyte reporter assay

与卵母细胞核成熟有关的事件已经很好地描述了。然而，对于细胞质中为受精和获得托蒂能做准备而发生的分子通路和过程，人们所知甚少。在卵母细胞成熟期间，基因表达的变化完全取决于母体信使RNA（mRNA）的转化和降解，而不是转录。因此，实施转化计划对建立卵母细胞发育能力以维持胚胎发育起着关键作用。本文是重点定义母体mRNA翻译程序的一部分，该程序发生在母体成熟和卵母细胞到卵母细胞的过渡阶段。在本方法论文中，提出了研究 体外 卵母细胞成熟期间目标mRNA翻译的调控策略。在这里，一个Ypet记者被融合到利益基因的3'未翻译区域（UTR），然后微注入卵母细胞与多基化mRNA编码为mCherry控制注射量。通过使用延时显微镜测量记者的积累，在卵母细胞成熟期间，在不同的过渡中计算翻译率。在这里，使用Ypet/插话-7（IL-7）-3'UTR记者为例，描述了卵母细胞分离和注射、延时记录和数据分析的协议。

21天大C57/BL6小鼠的被否定的阶段I-逮捕卵母细胞被注射了含有mRNA编码的记者组合，将Ypet记者融合到IL-7的3'UTR和mRNA编码mCherry。YFP和mCherry表达记录在39个卵母细胞中，其中30个已经成熟，9个在序言I中作为负控制被捕。三个成熟的卵母细胞被排除在分析之外，因为它们要么有延迟的GVBD（N+2），要么在录制过程中在盘子里移动（N=1）。 图3 显示了M樱桃和YFP表达在第一阶段和成熟?...

Watch this Scientific Journal Video about Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay at JoVE.com

Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay

该协议描述了记者在体外成熟期间研究单卵母细胞 mRNA 翻译的调节。

使用单卵母细胞记者测定在体外成熟期间的母体 mRNA 翻译程序

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that ...

此方法可用于识别和描述控制卵母细胞成熟期间翻译的调节元素。我们应用延时显微镜来评估记者在整个体外卵母细胞成熟过程中的积累。这准确地指出了在卵母细胞成熟期间发生转化激活或压抑的时间。首先，用26根针在毛囊壁上做一个小切口，小心地打开心角囊。使用口腔操作的玻璃移液器，将几层积积细胞分离出完整的 COC。使用较小的移液器，并通过重复管道机械地去污 COC。吸气的卵母细胞，并把它们放在一个培养皿与成熟介质补充一微摩尔的西洛斯塔米德。将菜放在孵化器中两小时，让卵母细胞从卵泡分离所引起的压力中恢复过来。将一根10厘米长的薄氧玻璃毛细管放在机械拉杆中，准备注射针头。使用加热灯丝以 45 度角弯曲针尖，以获得最佳注射效果。将20个基本卵母细胞收集介质的微滑液滴放在聚苯乙烯盘中，用轻质矿物油覆盖液滴。通过每微升 Ypet UTR 和 mCherry 各添加 12.5 微克，准备更大的记者组合。在零下80摄氏度下储存这些阿里报价。解冻后，以2万倍G将离心机切入两分钟，然后将其转移到新的微中转管。用大约0.5微升的记者混合加载注射针。将握管和注射针放入支架中，并将其放置在卵母细胞收集介质的液滴中。轻轻敲击注射针，使其贴在握住的移液器上，打开注射针。将卵母细胞放在基本收集介质的液滴中，并注射5到10个记者混合的皮质。用一种微摩尔西洛酰胺孵化卵母细胞和成熟介质16小时，使mCherry信号稳定下来。对于延时显微镜，为每个注射的记者准备一个培养皿，每个注射的记者有两个20微升的成熟介质滴，一个液滴与一个微摩尔西洛酰胺用于控制前兆I-逮捕的卵母细胞，一个没有西洛酰胺的液滴用于成熟的卵母细胞。用轻质矿物油盖住水滴，并将其放置在孵化器中。预孵化后，从孵化器中取出注射的卵母细胞，并在成熟介质中清洗四次，无氯酰胺。将一些卵母细胞保持在成熟介质中，其中一微摩尔西洛酰胺作为第一阶段卵母细胞控制组。将注射的卵母细胞转移到先前准备的延时显微镜盘上各自的液滴中。用封闭的玻璃移液器将卵母细胞聚集在一起，以防止它们在录制过程中移动。使用文本手稿中提到的参数，将盘子放在显微镜下，配备发光二极管消除系统和配备环境室的电动舞台，该室维护在 37 摄氏度和 5% 的二氧化碳中。通过单击应用，然后进行多维采集，输入延时实验的相应设置。选择第一个选项卡主选项卡，并选择延时、多个阶段位置和多个波长。选择"保存"选项卡以输入应保存实验的位置。然后选择选项卡延时输入时间点、持续时间和时间间隔数。选择禁忌阶段，打开光明场，并通过打开新窗口并选择获取、获取和显示实时来定位卵母细胞的位置。一旦卵母细胞被定位，切换回多维采集窗口，并按加设置卵母细胞的位置。选择选项卡波长，并为光明场、YFP 和 mCherry 设置三个不同的波长。调整 Ypet 和 mCherry 的曝光率，然后点击获取开始延时实验。为了分析Ypet三个主要的UTR翻译，执行两个区域测量每个卵母细胞，卵母细胞本身点击椭圆区域，和一个小区域周围的卵母细胞用于背景减法点击矩形区域。通过单击"打开日志"然后"日志数据"将区域测量数据导出到电子表格中。对于每个单独的卵母细胞和所有测量的时间点，从卵母细胞区域测量中分别减去YFP和mCherry波长的背景区域测量。在39个卵母细胞中记录了mCherry和YFP在序节I和成熟卵母细胞中的表达，其中30个已经成熟，9个在序言I中作为负控制被捕。通过将YFP信号除以前10个时间点的平均mCherry信号，将前一阶段I-逮捕和成熟卵母细胞的平均mCherry信号除以，纠正了注射量的单个YFP与mCherry比率。记者YFP的翻译率是通过曲线测量的YFP与mCherry比率在前一个零至两个小时，或8至10小时后，使用线性回归的西洛酰胺释放。记者的积累不遵循线性模式，如西洛酰胺释放后零至2小时和8至10小时之间的翻译率显著差异，表明卵母细胞肌成熟期间白细胞间7翻译激活。尝试此协议时，请确保在录制开始时曝光时间足够。请记住，如果在卵母细胞恢复期间激活翻译，信号可能会在时间过程中饱和。使用 YFP 的引金，PCR 可以确认 MRMA 的稳定性。为了确认翻译的变化，您可以使用带有抗体的西文印迹来对抗 V5 技术。

Research

JoVE Journal

Developmental Biology

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

科研

发育生物学

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

方法对角膜细胞的谱系追踪使用多色荧光记者小鼠

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Functional Cloning Using a Xenopus Oocyte Expression System

Functional Cloning Using a Xenopus Oocyte Expression System

功能性克隆使用<em&gt;爪</em&gt;卵母细胞表达系统

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

产妇血管重构的评估在怀孕期间在小鼠子宫

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

产妇基因产品的官能化操作使用<em&gt;体外</em斑马鱼&gt;卵母细胞成熟

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

使用电刺激人干细胞衍生的心肌细胞的成熟中Biowires

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

5-mC点印迹测定定量软骨细胞去分化的DNA甲基化水平<em&gt;体外</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation

使用mRNA电穿孔快速有效地表达Avian胚胎中的多种蛋白质

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. ...