种系线虫与胸苷模拟教育的细胞周期分析
Summary
本文描述了一种基于成像的方法, 可用于识别 S 相和分析种系中的细胞周期动力学. 此方法不需要转基因, 并且与免疫荧光染色兼容。
Abstract
真核细胞中的细胞周期分析经常利用染色体的形态、表达和/或定位所需的基因产品的各个阶段的细胞周期, 或合并核苷类似物。在 S 阶段, DNA 聚合酶将胸苷类似物 (如教育或 BrdU) 纳入染色体 DNA, 标记细胞进行分析。对于线虫, 核苷模拟教育在常规培养过程中被送入蠕虫, 并与免疫荧光技术兼容。种系是一种功能强大的模型系统, 用于研究信号通路、干细胞、减数分裂和细胞周期, 因为它是透明的、遗传的, 并且减数分裂前期和细胞分化/配子发生在线性装配样的时尚。这些特点使教育成为研究有丝分裂循环细胞和种系发展动态方面的一个很好的工具。该协议描述如何成功地准备教育细菌, 饲料他们到野生类型的线虫雌雄同体, 解剖双性性腺, 染色为教育纳入 DNA, 染色与抗体检测各种细胞周期和发展标记, 形象性腺和分析结果。该协议描述了测量 S 相指数、M 相指数、G2 持续时间、细胞周期持续时间、减数分裂率和减数分裂前期级数的方法和分析的变化。该方法可用于研究其他组织、阶段、遗传背景和生理条件下的细胞周期或细胞史。
Introduction
在动物发展中, 需要数以百计、数以千计、数以亿计的细胞分裂来形成成年有机体。细胞周期, 由 G1 (间隙)、S (合成)、G2 (间隙) 和 M (有丝分裂) 组成的细胞事件集定义了执行每个单元格除法的一系列事件。细胞周期是动态的, 最好是实时地得到赞赏, 这可能是技术上的困难。本协议中提供的技术允许一个人对静止图像进行细胞周期的相位和时间测量。
用核苷类似物 (如 5-乙炔-2 ‘-脱氧尿苷) 或 5-溴-2 ‘-脱氧尿苷 (BrdU) 进行标记, 是确定线虫线虫(线虫) 成人细胞周期动力学研究中 S 相的金标准。双性种系1,2,3,4,5。教育和 BrdU 几乎可以用于任何遗传背景, 因为它们不依赖任何基因结构。可视化 BrdU 需要苛刻的化学处理, 以暴露抗原的抗 BrdU 抗体染色, 这往往是不相容的评估其他细胞标记与其他抗体共同染色可视化。相比之下, 在轻度条件下, 通过单击化学进行可视化, 从而与抗体共染色6、7兼容。
标签的特异性是明确的, 因为原子核只在 S 阶段将胸苷 (5-乙炔-2 ‘-脱氧尿苷) 类似物纳入 DNA。可视化发生在固定组织中。该教育标签本身是无形的, 直到含有叠氮化物的染料或荧光基团与炔烃的铜催化单击化学8反应共价键。教育标签可以提供即时信息, 其中原子核是在 S 相, 使用短脉冲标记。教育还可以提供动态信息, 使用脉冲追逐或连续标记;例如, 在脉冲追逐实验中, 标签被稀释在每个细胞分裂或传播作为非分裂细胞进展通过开发。
线虫种系是一种强大的模型系统, 用于研究信号通路、干细胞、减数分裂和细胞周期。成人种系是一种极化装配线, 在远端发现了干细胞, 随后通过减数分裂前期进入和级数, 与配子的分期相协调 (图 1)。在近端, 卵母细胞成熟, 是排卵和受精和开始胚胎发生在子宫9,10,11。远端尖端细胞附近的约20个细胞直径长的区域, 包括有丝分裂循环种系茎、祖细胞和减数分裂的 S 相细胞, 而不是减数分裂前期的细胞, 被称为祖区2,4,9,12. 细胞膜在远端种系核之间不完全分离, 但祖细胞在很大程度上独立进行有丝分裂细胞循环。年轻成人雌雄同体种系祖区细胞中有丝分裂细胞周期的中位数为 6.5 h;G1 期短或不存在, 平静未观察到1、2、13。种系干细胞分化发生的主要原因是直接分化, 因此缺乏4的过境放大。在粗线期阶段的分化过程中, 大约4的5核不会形成卵母细胞, 而是通过细胞凋亡, 通过将胞质含量捐献给发育中的卵子12、14 , 作为护士体细胞。,15。
除了用核苷类似物标记 S 相的细胞外, 还可以使用抗体染色识别有丝分裂和减数分裂中的细胞。有丝分裂中的细胞核是免疫的抗磷组蛋白 H3 (Ser10) 抗体 (称为 pH3)7,16。减数分裂中的细胞核是免疫 anti-HIM-3 抗体 (减数分裂染色体轴蛋白)17。祖区的细胞核可以通过缺乏 HIM-3、nucleoplasmic REC-818的存在或 WAPL-119的存在来识别。WAPL-1 强度在体细胞性腺中最高, 祖区高, 早期减数分裂 prophases19。几个细胞周期测量是可能的, 在协议中的一些变化: I) 在 s 相和测量 s 相指数中识别核;II) 确定 m 相核, 测量 m 相指数;III) 确定核是否在有丝分裂或减数分裂的 S 阶段;IV) 测量 G2 的持续时间;V) 测量 G2+M+G1 阶段的 duartion;VI) 测量减数分裂的进入率;VII) 估计减数分裂的进展率。
一个可以做多细胞周期测量从几个类型的湿实验室实验。下面的协议描述了30分钟脉冲标记喂养C. 线虫成人雌雄同体与教育标记细菌和联合标记 M 相细胞通过染色与 anti-pH3 抗体和祖细胞的染色 anti-WAPL-1 抗体。只有在教育饲料的持续时间 (步骤 2.5), 使用的抗体类型 (步骤 5) 和分析 (步骤 8.3) 的变化在额外的测量需要。
Protocol
Representative Results
Discussion
教育标记细菌的制备 (步骤 1) 对此协议至关重要, 也是解决问题的第一步。野生型年轻成人雌雄同体标签非常可靠地在一个4小时的教育脉冲, 使这对每一批新的教育标记细菌是一个有用的控制。此外, 完整的教育标记细菌进入肠道 (在老动物或某些咽/磨床缺陷突变体) 将标签与点击化学和显示为明亮的长方形斑点在肠道。用于标记雌雄同体的另一种技术是在3的高浓度 (1 mM) 中使用 …
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢 MG1693 的大肠杆菌库存中心;Wormbase;线虫遗传学中心, 由国立卫生研究院的研究基础设施项目 (P40OD010440) 资助, 用于菌株;统计咨询;陈爱萍丰试剂;Scharf, 安德烈. 施耐德, 桑迪普, 和约翰的培训, 建议, 支持和有益的讨论;Kornfeld 和施德实验室对这份手稿的反馈意见。这项工作由国家卫生研究院 (R01 AG02656106A1 到 KK、R01 GM100756 到 TS) 和国家科学基金会 predoctoral 奖学金 [DGE-1143954 和 DGE-1745038] 部分支持。无论是国家卫生研究院还是国家科学基金会, 在设计研究、收集、分析和解释数据方面, 都没有任何作用, 也没有写手稿。
Materials
| E. coli MG1693 | Coli Genetic Stock Center | 6411 | grows fine in standard unsupplemented LB |
| E. coli OP50 | Caenorhabditis Genetics Center | OP50 | |
| Click-iT EdU Alexa Fluor 488 Imaging Kit | Thermo Fisher Scientific | C10337 | |
| 5-Ethynyl-2′-deoxyuridine | Sigma | 900584-50MG | or use EdU provided in kit |
| Glucose | Sigma | D9434-500G | D-(+)-Dextrose |
| Thiamine (Vitamin B1) | Sigma | T4625-5G | Reagent Grade |
| Thymidine | Sigma | T1895-1G | BioReagent |
| Magnesium sulfate heptahydrate | Sigma | M1880-1KG | MgSO4, Reagent Grade |
| Sodium Phosphate, dibasic, anhydrous | Fisher | BP332-500G | Na2HPO4 |
| Potassium Phosphate, monobasic | Sigma | P5379-500G | KH2PO4 |
| Ammonium Chloride | Sigma | A4514-500G | NH4Cl, Reagent Plus |
| Bacteriological Agar | US Biological | C13071058 | |
| Calcium Chloride dihydrate | Sigma | C3881-500G | CaCl |
| LB Broth (Miller) | Sigma | L3522-1KG | Used at 25g/L |
| Levamisole | Sigma | L9756-5G | 0.241g/10ml |
| Phosphate buffered saline | Calbiochem Omnipur | 6506 | homemade PBS works just as well |
| Tween-20 | Sigma | P1379-500ML | |
| 16% Paraformaldehyde, EM-grade ampules | Electron Microscopy Sciences | 15710 | 10ml ampules |
| 100% methanol | Thermo Fisher Scientific | A454-1L | Gold-label methanol is critical for proper morphology with certain antibodies |
| Goat Serum | Gibco | 16210-072 | Lot 1671330 |
| rabbit-anti-WAPL-1 | Novus biologicals | 49300002 | Lot G3048-179A02, used at 1:2000 |
| mouse-anti-pH3 clone 3H10 | Millipore | 05-806 | Lot#2680533, used at 1:500 |
| goat-anti-rabbit IgG-conjugated Alexa Fluor 594 | Invitrogen | A11012 | Lot 1256147, used at 1:400 |
| goat-anti-mouse IgG-conjugated Alexa Fluor 647 | Invitrogen | A21236 | Lot 1511347, used at 1:400 |
| Vectashield antifade mounting medium containing 4',6-Diamidino-2-Phenylindole Dihydrochloride (DAPI) | Vector Laboratories | H-1200 | mounting medium without DAPI can be used instead, following a separate DAPI incubation |
| nail polish | Wet n Wild | DTC450B | any clear nail polish should work |
| S-medium | various | see wormbook.org for protocol | |
| M9 buffer | various | see wormbook.org for protocol | |
| M9 agar | various | same recipe as M9 buffer, but add 1.7% agar | |
| Nematode Growth Medium | various | see wormbook.org for protocol | |
| dissecting watch glass | Carolina Biological | 42300 | |
| Parafilm laboratory film | Pechiney Plastic Packaging | PM-996 | 4 inch wide laboratory film |
| petri dishes | 60 mm diameter | ||
| Long glass Pasteur pipettes | |||
| 1ml centrifuge tubes | MidSci Avant | 2926 | |
| Tips | |||
| Serological pipettes | |||
| 500 mL Erlenmyer flask | |||
| Aluminum foil | |||
| 25G 5/8” needles | BD PrecisionGlide | 305122 | |
| 5ml glass centrifuge tube | Pyrex | ||
| Borosilicate glass tubes 1ml | |||
| glass slides | |||
| no 1 coverslips 22 x 40 mm | no 1.5 may work, also | ||
| 37 °C Shaker incubator | |||
| Tabletop Centrifuge | |||
| Clinical Centrifuge | IEC | 428 | with 6 swinging bucket rotor |
| Mini Centrifuge | |||
| 20 °C incubator | |||
| 4 °C refrigerator | |||
| -20 °C freezer | |||
| Observer Z1 microscope | Zeiss | ||
| Plan Apo 63X 1.4 oil-immersion objective lens | Zeiss | ||
| Ultraview Vox spinning disc confocal system | PerkinElmer | Nikon spinning disc confocal system works very well, also, as described here: http://wucci.wustl.edu/Facilities/Light-Microscopy |
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