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
1Center for Reproductive Sciences,University of California at San Francisco, 2Department of Obstetrics Gynecology and Reproductive Sciences,University of California at San Francisco, 3Present affiliation: Laboratoire de Biologie du Développement-Institut de Biologie Paris Seine, LBD-IBPS,Sorbonne Université
Summary
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。
Abstract
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。
Introduction
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。
Protocol
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 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。 2. 为 Ypet-3′ UTR ?…
Representative Results
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?…
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| Preparation of media | |||
| Bovine Serum Albumin Powder Bioxtra | Sigma-Aldrich | SIAL-A3311 | |
| Cilostamide | EMD Millipore | 231085 | |
| MEM alpha | Gibco | 12561-056 | |
| Minimum Essential Medium Eagle | Sigma-Aldrich | M2645 | |
| Penicillin-Streptomycin 100x Solution, Sterile Filtered | Genesee Scientific Corporation (GenClone) | 25-512 | |
| Sodium Bicarbonate | JT-Baker | 3506-1 | |
| Sodium Pyruvate | Gibco | 11360-070 | |
| Ultrapure distilled water | Invitrogen | 10977-015 | |
| Preparation of mRNA encoding YFP/3' UTR and mCherry | |||
| Agarose | Apex Biomedical | 20-102QD | |
| Carbenicillin disodium salt | Sigma-Aldrich | C1389-1G | |
| Choo-Choo Cloning Kit | McLab | CCK-20 | |
| CutSmart Buffer (10x) | New England Biolabs | B7204 | |
| DNA loading dye (6x) | Thermo Scientific | R0611 | |
| dNTP Solution | New England Biolabs | N0447S | |
| DpnI | New England Biolabs | R0176 | |
| GeneRuler 1 kb DNA ladder | Thermo Fisher | SM1333 | |
| LB Agar Plates with 100 µg/mL Carbenicillin, Teknova | Teknova | L1010 | |
| LB Medium (Capsules) | MP Biomedicals | 3002-021 | |
| MEGAclear Transcription Clean-Up Kit | Life Technologies | AM1908 | |
| MfeI-HF restriction enzyme | New England Biolabs | R3589 | |
| mMESSAGE mMACHINE T7 Transcription Kit | Invitrogen | AM1344 | |
| Phusion High Fidelity DNA polymerase | New England Biolabs | M0530 | |
| Poly(A) Tailing kit | Invitrogen | AM1350 | |
| QIAprep Spin Miniprep Kit | Qiagen | 27106 | |
| QIAquick Gel Extraction Kit | Qiagen | 28704 | |
| S.O.C. medium | Thermo Fisher | 15544034 | |
| TAE buffer | Apex Biomedical | 20-193 | |
| Ultrapure Ethidium Bromide Solution | Life Technologies | 15585011 | |
| Oocyte collection | |||
| Aspirator tube assembly for calibrated micro-pipettes | Sigma-Aldrich | A5177-5EA | |
| Calibrated micro-pipettes | Drummond Scientific Company | 2-000-025 | |
| PMSG- 5000 | Mybiosource | MBS142665 | |
| PrecisionGlide Needle 26 G x 1/2 | BD | 305111 | |
| Syringe 1 ml | BD | 309659 | |
| Oocyte micro-injection | |||
| 35 mm Dish | No. 0 Coverslip | 20 mm Glass Diameter | Uncoated | MatTek | P35G-0-20-C | For time-lapse microscopy |
| Borosilicate glass with filament | Sutter Instrument | BF100-78-10 | |
| Oil for Embryo Culture | Irvine Scientific | 9305 | |
| Petri Dish | Falcon | 351006 | For micro-injection |
| Tissue Culture Dish | Falcon | 353001 | For oocyte incubation |
| VacuTip Holding Capillary | Eppendorf | 5195000036 | |
| Software | |||
| Biorender | BioRender | Preparation of Figure 1S | |
| MetaMorph, version 7.8.13.0 | Molecular Devices | For time-lapse microscopy, analysis of 3' UTR translation |
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Citer Cet Article
Costermans, N. G. J., Daldello, E. M., Marathe, R. J., Conti, M. Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay. J. Vis. Exp. (172), e62041, doi:10.3791/62041 (2021).