文化早期鸡胚体外（新文化）的方法
Summary
本视频演示了新的文化，鸡胚培养24小时以外的鸡蛋的方法。这种方法使研究的早期发展（原始连胜，以14索姆），鼠标E7 – 9对应一个时期。这项技术的应用，包括电，原位杂交和免疫组化。
Abstract
鸡胚是一个有价值的工具，在早期胚胎发育的研究。其透明度，可获得性和易于操纵，它为研究大脑，神经管，体节和心脏原基的形成和图案的理想工具。鸡胚培养的应用包括电的DNA或RNA的结构，以原位杂交和免疫组织化学分析基因的功能，生长因子，如涂珠FGFs和BMPs的移植，以及整装。本视频演示了在鸡胚培养的不同步骤，首先，胚胎是在生理盐水explanted。然后，胚胎中心的玻璃环上。周围的胚胎膜沿环的墙壁被取消。环，然后放置在文化菜含有白蛋白池。是密封的培养皿中，并放置在潮湿的腔，胚胎培养24小时。最后，胚胎是从环中删除，固定，并为进一步的应用处理。还介绍了故障排除指南。
Protocol
第1部分：台成立一股潮湿室是放置在塑料商会Kimwipe / DDH 2 O的准备。 一个猎鹰管收集白蛋白，文化，戒指，表面玻璃和废物处置的菜都放在替补席上。 耐热盘充满生理盐水1.4升（见注释[A]）。 第2部分：胚胎在盐水explanted 鸡蛋是从孵化器中删除后16小时（第4阶段）。鸡蛋是由攻壳钳opene。壳牌件被删除。 薄白蛋白收集猎鹰管。?…
Discussion
一个广泛的应用，从增长因素含珠 3，整个装载在原位杂交和整装免疫组织化学 4移植，可用于新的培养方法2。超过24小时期间的文化，使胚胎发育的连续监测中的应用，如时间的推移细胞运动分析5或绿色荧光蛋白含有电穿孔结构6监测。
Acknowledgements
这项工作是支持的玛格丽特M. Alkek基金会RHF。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Eggs | Animal | Charles River Laboratories | Premium Fertile | |
| Stereomicroscope | Microscope | Leica Microsystems | MZ9.5 or similar | |
| Marsh Automatic Incubator | Tool | Lyon | RX | |
| Hybridization Incubator | Tool | Robbins Scientific | M1000 | |
| Pyrex dish (2) | Tool | |||
| Watchmaker’s glass 50mm | Tool | VWR | 66112-060 | |
| Glass rings | Tool | Physical Plant facility | cut 4 mm thick sections of glass tubing (27 mm outer diam, 25 mm inner diam). Do not fine polish. | |
| Curved Forceps (1) | Surgery | Electron Microscopy Sciences | 72991-4C | |
| Forceps (2) | Surgery | Fine Science Tools | 11002-13 | blunt ended using sharpening Stone and 100ul mineral oil |
| Sharpening Stone Dan’s Black Arkansas | Surgery | Electron Microscopy Sciences | 62082-00 | |
| Fine scissors | Surgery | Fine Science Tools | 14161-10 | |
| Plastic dishes | Tool | Falcon | 353001 | |
| Rubber Bulb | Tool | Electron Microscopy Sciences | 70980 | |
| Pasteur Capillary Pipette | Tool | Electron Microscopy Sciences | 70950-12 | round edge under flame |
| Microcapillary tube | Surgery | Sigma | P1049-1PAK | Pull using vertical micropipette puller; blunt end with fine forceps |
| Microdissecting knife | Surgery | Fine Science Tools | 10056-12 | Use to puncture cavities prior to in situ hybridization |
| Minuten pins 0.2mm diam | Surgery | Fine Science Tools | 26002-20 | |
| Sylgard 184 Silicon Elastomer Curing Agent and Base | Reagent | Dow Corning | 0001986475 | Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C |
| Diethylpyrocarbonate (depc) | Reagent | Acros Organics | 10025025 | Add 1ml depc to 1l PBS; shake; autoclave |
References
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- Voiculescu, O., Papanayotou, C., Stern, C. D. Spatially and temporally controlled electroporation of early chick embryos. Nature Protoc. 3, 419-426 (2008).
Cite This Article
Psychoyos, D., Finnell, R. Method for Culture of Early Chick Embryos ex vivo (New Culture). J. Vis. Exp. (20), e903, doi:10.3791/903 (2008).