联体斑马鱼胚胎一代发展囊胚的手术融合
Summary
This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.
Abstract
不同的遗传背景的两种动物的手术间生态创建了一个独特的场景来研究细胞内在与感兴趣的候选基因，细胞的迁徙行为，并在不同的遗传设置分泌信号，细胞外源性的角色。由于联体动物有着共同的循环，从一个动物的血液或血源性因素将与其合作伙伴，反之亦然交换。因此，从一个遗传背景的细胞和分子因素可在第二遗传背景的情况下进行研究。成年小鼠的联体已被广泛用于研究衰老，癌症，糖尿病，肥胖，和大脑的发育。最近，斑马鱼胚胎的联体已被用于研究造血发育生物学。与此相反的小鼠，斑马鱼胚胎的透明特性允许细胞的直接可视化的背景下连体，使其成为调查为f的独特而强大的方法undamental细胞和分子机制。这种技术的效用，但是，通过用于产生连体斑马鱼胚胎陡峭的学习曲线限定。这个协议提供了关于如何手术融合不同的遗传背景两个斑马鱼胚胎的囊胚调查感兴趣的候选基因的作用的工序一步方法。此外，联体斑马鱼的胚胎是耐受热冲击，使得基因表达可能的时间控制。该方法不需要复杂的设置和对胚胎发育过程中研究体内细胞迁移，命运说明书中，并分化广阔的应用领域。
Introduction
野生型和转基因动物的遗传马赛克（嵌合体）的创建是调查细胞内在与候选基因1-6的细胞外功能的完善和经典的策略。在斑马鱼囊胚移植已被广泛使用，以产生用于细胞自治7-9的研究嵌合胚胎。取决于感兴趣的组织，但是，它可以是具有挑战性的供体细胞可预测定位到期望的组织（ 例如，血液）1-3，7-9。鼠标遗传学家早就采用连体手术的方法来产生连体生物与共享循环10-14。由于联体动物都有一个共同的血液，即来自一种动物具有不同遗传背景的其他动物的细胞的细胞之间的相互作用，可以研究10-16。近日，Karima Kissa小组优雅证明CRE的能力连体吃斑马鱼胚胎，然后用这个系统来研究造血干祖细胞（HSPC）的迁移15。此外，斑马鱼联体最近用于研究内皮钙5的造血干细胞（HSC）的出现和迁移，16的作用和斑马鱼发育17中，研究中的HSPC利基基质细胞的作用。
不像小鼠，斑马鱼的胚胎是透明的，允许联体发育过程中的细胞的直接可视化，使得系统唯一地强大。斑马鱼中的联体的效用，然而，由一个陡峭的学习曲线，并在胚胎细腻手术连体限制可以没有详细的说明和视觉示范技术上的挑战。该协议的目标是提供一套明确的，一步一步的指示陪如何产生连体斑马鱼胚胎为研究基于视频的教程颞，细胞 – 本征的，或者一个候选基因（s）的显影囊胚的手术融合的细胞外功能。重要的改进和提高parabiont生存和新的实验性应用程序的其他建议包括在内。
Protocol
Representative Results
Discussion
联体融合已调查在成年鼠模型和鸡胚10-14候选基因的细胞功能的有力工具。最近，囊胚融合方法已用于产生连体斑马鱼胚胎15所述。在本协议中，基于视频的教程用于演示和更好地描述的方法为，以研究在造血发育感兴趣的候选基因的时空，细胞 – 本征的，和细胞外的角色创建不同遗传背景的连体斑马鱼的胚胎超越。
本文中所描述的方法，最近被用于跟踪的HSC…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We thank Julie R. Perlin for helpful comments on the manuscript. D.I.S. is supported by grants from the American Society of Hematology, the Cooley’s Anemia Foundation, and the NIH (K01DK085217 and R03DK100672). E.J.H. is a Howard Hughes Medical Institute Fellow of the Helen Hay Whitney Foundation. B.L. is a Howard Hughes Medical Institute Medical Research Fellow. B.W.B. is supported by an Irvington Fellowship from the Cancer Research Institute and a Young Investigator Award from the Conquer Cancer Foundation of ASCO. L.I.Z. is supported by grants from the NIH (R01CA103846, P01HL032262, and R01HL04880), Taub Foundation for MDS Research, Harvard Stem Cell Institute, and is an investigator of the Howard Hughes Medical Institute.
Materials
| Methyl cellulose | Sigma | M0387 |
| Individual components for E3/HCR: | For 1L: 14,61g NaCl, 0,63g KCl, 2,43g CaCl2-2H2O, 1,99g MgSO4 | |
| NaCl (Sodium chloride) | Sigma-Aldrich | S9888 |
| KCl (Potassium chloride) | Sigma-Aldrich | P9541 |
| CaCl2 (Calcium chloride dihydrate) | Sigma-Aldrich | 223506 |
| MgSO4 (Magnessium sulfate heptahydrate) | Sigma-Aldrich | 230391 |
| Hepes (1M ) buffer solution | ThermoFisher | 15630-080 |
| Name | Company | Catalog Number |
| Antibiotics: | ||
| Pen/Strep | gibco by Life technologies | 15140-120 |
| Ampicilin sodium salt | Sigma Life Science | A0166 |
| Kanamycin sulfate from Streptomyces kanamyceticus | Sigma Life Science | K1377 |
| Name of Reagent/ Equipment | Company | Catalog Number |
| 50 mg/ml Pronase from Streptomyces griseus | Roche | 11459643001 |
| capillary glass used for needles (Capillary Glass & Filaments) | Sutter Instrument | ITEM#: BF 100-50-10 |
| Teasing needles with wooden handles | Fisher Scientific | S07894 |
| Glass Pasteur pipettes | Fisherbrand | 13-678-20A |
| 10 mL pipette pump (green) (Pipette Pump Pipettor) | Novatech International | F37898-0000 |
| 100 mm diameter/ 20 mm deep plastic petri dishes (PETRI DISH, 100/20 MM, PS, CLEAR, WITH VENTS, HEAVY DESIGN, 15 PCS./BAG ) |
Greiner Bio-one | 664102 |
| Dextran, Cascade Blue, 10,000 MW, Anionic, Lysine Fixable | ThermoFisher | D-1976 |
| PTU. working stock is 0.003% (50X is 0.15%). for 500ml, 0.75 g N-Phenylthiourea | Sigma-Aldrich | P7629 |
| Tricaine (powder) (Tricaine Methanesulfonato, Tricaine-S) | Western Chemical Inc | MS 222 |
| LMP agarose (Ultrapure LMP agarose) | Invitrogen | 16520100 |
| plastic transfer pipette (just the wide ended one I think) | Fisherbrand | 137115AM |
| Glass-bottom 6-well plates used for imaging | MatTek | P06G1.5-20-F |
| plastic western gel loading tip fixed on the end of a wood-handled dissecting needle (GELoader tips) | Eppendorf | 22351656 |
| glass cover slips, slides and vacuum grease if mounting for an upright microscope: | ||
| Vaccum grease ( Dow Corning® high-vacuum silicone grease colorless, weight 5.3 oz (tube) ) |
Dow Corning | Z273554 |
| Glass cover slips | Corning Life Sciences | 2960-244 |
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