移植嵌合斑马鱼胚胎生成
Summary
在囊胚或原肠胚阶段有针对性的嵌合体斑马鱼的胚胎移植产生由的一步一步的指导。
Abstract
使用到复杂的发育过程中获得的洞察力最有力的工具之一是嵌合体胚胎的分析。一种幻想被定义为一个有机体，它包含从一个以上的动物细胞;马赛克是一个从多个基因型的细胞混合，通常是野生型和突变型嵌合型。在斑马鱼中，嵌合体可以随时到主机胚胎细胞在适当的萌芽阶段，从捐赠者胚胎移植。注射到一个细胞阶段的胚胎，一个宗族标记，如荧光染料标记的供体细胞的生成。删除标记的供体细胞胚胎从供体和引入到未标记的主机使用胚胎石油控制的玻璃吸管，装在一个化合物或解剖显微镜。供体细胞可以在某些情况下，一个特定的地区或发展囊胚或原肠胚阶段主机胚胎组织有针对性地选择根据既定的命运地图的主机胚胎移植部位。
Protocol
Discussion
与移植可用于生产有针对性的嵌合体的缓解是斑马鱼为脊椎动物模式的大国之一。上面没有说明这个协议的修改，允许母体基因的功能分析与合子发展中的重要作用的基因。由于这些突变的鱼无法生存，直到成年后，有必要转移到其他野生型主机胚胎生殖细胞突变，突变的细胞的“生殖克隆”。生成生殖马赛克涉及移植的原始野生型主机胚胎生殖细胞突变的捐赠。与所有其他在胚胎细胞谱系，生?…
Acknowledgements
在写这篇文章很有帮助输入Moens实验室的成员。香港是由美国国立卫生研究院/ NICHD的授予＃5R01HD037909 – 08支持的博士后研究员。煤层气是与霍华德休斯医学研究所研究员。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Injection rig | Applied Scientific Instrumentation | MPPI-3 | Foot pedal can be ordered separately | |
| Pronase | Sigma | P5147 | “protease type XIV” | |
| Pen-Strep | Sigma | P4458 | 100x stock | |
| Methyl cellulose | Sigma | M-0387 | ||
| Fluorescent dextrans | Molecular Probes/ Invitrogen | various | Eg. lysine-fixable rhodamine dextran (D7162) | |
| Injection pipettes (1.2mmx0.94mmx10cm) | Sutter | BF120-94-10 | ||
| Transplant pipette option 1 | World Precision Instruments | TW100-4 | ||
| Transplant pipette option 2 | VWR | #53508-400 | ||
| micromanipulator | Narishige | UMJ-3FC (?) | ||
| Micromanipulator magnetic stand | Narishige | GJ-1 | ||
| Transplant apparatus | Sutter Instrument Co. | MI-10010 | ||
| Fine micromanipulator | Narishige | MMO-203 | ||
| Depression slides | Fisher | 12560A | ||
| Agar molds for injection and transplants | AL-AN Mfg, Inc. |
Reagents: Recipes for many of the reagents used for these procedures are found in The Zebrafish Book, which is available in full online at http://zfin.org/zf_info/zfbook/zfbk.html. Recipes are provided there for preparation of Fish Water, Embryo Medium with and without antibiotics, pronase for dechorionating, fluorescent dextrans, and methyl cellulose.
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