由于硬的蛋壳和软的胚胎,操纵鳉胚胎是比在斑马鱼中更多地参与。该视频显示一步一步的程序,包括dechorionation如何操作鳉胚胎,在琼脂糖凝胶成像和细胞移植嵌合体的生产安装。这些程序是必不可少的,在实验室中使用鳉和斑马鱼为脊椎动物的基因组功能基因清扫充分利用其互补性的特点。
青鳉是一个小产蛋的淡水鱼类,允许遗传和胚胎学分析,是在表型驱动的全基因组突变的屏幕进行了1生物体的脊椎动物模型之一。鳉和斑马鱼之间的相关基因的功能重叠的分歧使得新颖,品种单一,从而鳉和斑马鱼是脊椎动物的基因组功能基因清扫互补难以辨认的表型鉴定。青鳉胚胎,如dechorionation,安装成像和细胞移植的胚胎,操纵鳉和斑马鱼在实验室工作的主要程序。细胞移植研究细胞鳉突变的自主权。嵌合体产生的未标记的收件人胚胎移植到从捐赠的胚胎标记细胞。供体细胞可以移植到特定领域基于命运的地图3上,使移植细胞克隆可以集成在开发过程中的利益的组织中的收件人胚胎。由于硬的蛋壳和软的胚胎,操纵鳉胚胎是比在斑马鱼中更多地参与。在这个视频中,我们展示操纵鳉胚胎的详细程序。
在这个视频中,我们将演示如何dechorionate开展鳉胚胎细胞transplanataion。这是一个强大的技术,生产用于在开发过程中的基因/蛋白功能的研究,以及阐明了有问题的基因/蛋白质的自主权的嵌合胚胎。青鳉是一个有用的脊椎动物模型系统行之有效的命运地图和其透明度贷款,它们在体内的实时成像,由于这种技术。移植可结合显微注射(如在陪同朱庇特议定书“青鳉胚胎显微注射”所述),因此可以很容易地移植细胞的行为观察。
The authors have nothing to disclose.
这项工作是支持由授予的MRC到M FS。
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Embryo medium | Reagent | Made in-house | N/A | 200ml 50X stock solution, 1ml 1%methyleneblue in H2O/10 liter RO water (50X stock solution: NaCl 14.7g, KCl 0.6g, CaCl2 2H2O 2.4g, MgSO4 7H2O 4.0g/1 litre RO water.) |
Micro-dissecting forceps | Tools | 55 INOX A.DUMONT&FILS | N/A | Very sharp fine tips for removal of chorion. |
Fine waterproof sandpaper – p2000 grit size | Tools | Hermes Abrasives Ltd. | N/A | Very fine grit size for rolling/cleaning embryos with chorions. |
3% methylcellulose | Reagent | Sigma | M 0512 | High viscosity for holding embryos in place during transplantation. |
1X BSS | Reagent | Made in-house | N/A | 20XBSS: 130g NaCl, 8g KCl, 4g MgSO4.7H2O, 4g CaCl2.2H2O, and 10mg Phenol Red in 1L MilliQ water and autoclave; 500mM Hepes in MilliQ water autoclaved. Add 25ml 20XBSS and 15ml 500ml Hepes pH7.0, fill up to 500ml and filter sterilize before use. |
Penicillin-streptomycin | Reagent | Gibco | 15140-122 | Added to BSS when incubating dechorionated embryos. |
Pronase | Reagent | Calbiochem | 53702 | For dechorionation. |
Hatching enzyme | Reagent | Made in-house | N/A | For dechorionation. |
Tricaine mesilate (TMS) | Reagent | Sigma | A-5040 | 400mg tricaine in 97.9ml distilled water and 2.1ml 1M Tris, pH adjusted to 7; 4.2ml of this solution in 100ml clean tank water. |
3% ultra-low gelling temp. agarose | Reagent | Sigma | A-2576 | Type IX-A. Used during embedding of embryos for observation. |
Petri dish culture chamber | Tool | Iwaki, Asahi Techno Glass Corp. | 11-004-008 | Iwaki glass base dish 35mm with 12mm window. Used during embedding of embryos for observation. |
Micromanipulator | Equipment | Narishige | N/A | Model MN151. For removal and insertion of cells during transplantation. |
Borosilicate glass capillaries | Tool | Harvard Apparatus | GC100-10T | For making transplantation needles. |
Flaming/Brown micropipette puller | Equipment | Sutter Instrument Co. | Model P-97 | For making transplantation needles. |