创建定义的气体环境研究缺氧对<em> C。线虫</em
Summary
本文详细介绍了如何使用连续流缺氧商会产生与定义的O浓度的大气<sub> 2</sub>了解生物反应下降Ø<sub> 2</sub>。该系统易于安装和维护，并具有足够的灵活性，以适应广泛的O<sub> 2</sub>浓度和模型系统
Abstract
氧气是必不可少的，所有的多细胞动物的生存与一个已知的例外1。降低O 2的可用性（缺氧）中出现的疾病，发育正常或在2-5环境条件的变化状态。了解细胞信号途径参与缺氧反应可以分为治疗策略提供新的见解，对不同的人类疾病，中风，癌症。已阻碍了这一目标，至少有一部分，控制低氧暴露在模式生物基因服从相关的技术困难。
线虫作为模式生物缺氧反应的研究，因为它非常适合容易培养和基因操纵。此外，它可能没有因为C.影响因子的细胞反应到具体缺氧O 2的浓度研究线虫获得O 2的 （和其他气体扩散），而不是一个促进呼吸道系统6。 在 C已知缺氧反应涉及的因素是保守线虫 。缺氧的实际响应依赖于特定的O 2浓度可用。 在 C 线虫 ，中度缺氧暴露引出一个主要介导的HIF-1的转录反应，高度保守的缺氧诱导转录因子6-9，C。线虫胚胎需要HIF-1在5,000-20,000 ppm的生存Ø2 7,10 。缺氧是一个“O 2的比正常少”的总称。常氧（O 2的正常），也可以是很难界定。我们普遍认为室内空气，这是21万ppm的O 2的是常氧。然而，它已被证明是C。线虫有行为偏好O 2的浓度从5-12％（50,000-120,000 ppm的氧 ）11。在Larvae和成年人，HIF-1的行为，以防止缺氧诱导滞育的5000 PPMØ2 12。然而，HIF-1不玩作用，在低浓度氧 （缺氧，<10 ppm的O 2的操作性定义）13。在缺氧，C。线虫进入一种假死的可逆状态，在所有显微镜下观察到的活动停止10。事实上，出现不同的生理反应，强调在不同条件下的缺氧O 2的浓度在实验控制的重要性。
在这里，我们提出了一个可靠和可重复性缺氧条件下产生的O 2浓度定义的环境商会的建设和实施方法。在不断流的方法，确保迅速达到平衡室，并增加了系统的稳定性。此外，透明度和无障碍商会让动物受到缺氧的直接可视化。我们进一步证明有效的方法收获C.线虫样本迅速缺氧暴露后，这是必须遵守许多的迅速反转的变化发生在缺氧10,14。这种方法提供了一个基本的基础，可以很容易地修改个别实验室的需求，包括不同的模型系统和各种气体。
Protocol
1。建设环境试验箱选择最小的室为您的项目范围内的合理量。商会必须由气体（O）的防渗材料。高硼硅结晶菜，Anaeropack盒，或大型铸铁压克力盒（Ellard仪表），都可以使用。我们已经发现，9 50毫米的钢板可以在100×50 Kimex结晶菜适合。玻璃板可以用作耐热结晶菜肴的盖子。 在选定的腔钻一个洞，适合塑料软管倒钩接头（科尔帕默）男性露儿。配件可以保证管件或用环氧树…
Discussion
这种方法提出了一个缺氧的环境，允许精确的氧气浓度维持在实验室环境建设的战略。这些商会提供了一个简单的方法暴露生物体特定的低浓度的O 2和监测的分子和生理输出。环境试验箱组装实验室，而不是商业购买，因此可以进行修改，以满足实验的需要。
这种方法的一个明显优势是连续流设计。这消除了通常与维护商会O 2的低浓度时，外部的氧?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢米勒实验室成员讨论和批判性阅读的手稿。这项工作是支持的弥敦道冲击卓越中心由新研究者奖老化DLM和国家机构的健康奖R00为AG030550到DLM的生物学基础。
Materials
| Name of the reagent | Company | Catalogue number |
| Tubing (FEP and PTFE) | Cole Parmer Tygon |
YO-95821-00 (1/8″ FEP) 06605-27 (1/16 x 1/8″ PTFE)’ R-3603 |
| Compression fittings | Seattle Fluid Systems | 06363-58 (M. coupler 1/16″) 06363-62 (F. coupler 1/16″) 06363-60 (M. coupler 1/8″) 06363-61 (F. coupler 1/8″) |
| Flow tube | Aalborg | PMR3-010073 (3 output) PMR1-013520 (1 output) |
| Mass flow controller | Sierra Instruments | 810S-L-DR-2-OV1-SK1-V1-S1 (Mass Trak) C100L-DD-2-OV1-SV1-PV2-V1-SO-C10 (Smart Trak 2) |
| Compressed gas tank | AirGas | Made to order |
| Plastic male Luer to hose barb fittings | Cole Parmer | 45505-41 (500 series 1/16″) |
| Cast acrylic boxes | Ellard Instrumentation | Made to order |
| Pipe fittings (Brass or stainless steel) | Seattle Fluid Systems | B-402-1 (1/4″ nut) B-200-3 (1/8″ union tee) B-400-set (1/4″ ferrules) B-QM2-B1-200 (QM Body QC) B-200-1-2 (1/8 x 1/8″ male conn) |
| Dow Corning Vacuum Grease | Sigma-Aldrich | Z273554 |
| AnaeroPack box | Misubishi Gas Chemical Company | R684004 (0.4 liter) R685025 (2.5 liter) R685070 (7.0 liter) |
| Pyrex gas wash bottle | Sigma-Aldrich | CLS31770500C (500 mL) CLS31770250C (250 mL) CLS31770125C (125 mL) |
| Palmitic acid | Sigma-Aldrich | P0500 |
| Goat anti-mouse IgG-horseradish peroxidase | Southern Biotechnology Associates | 1032-05 |
| SuperSignal West Pico Chemiluminsecent Substrate | Pierce Chemical | 34077 |
| 100 x 50 glass crystallization dishes | Kimax Kimble | 23000 |
References
- Danovaro, R. The first metazoa living in permanently anoxic conditions. BMC Biology. 8, 30 (2010).
- Birner, P. Overexpression of Hypoxia-inducible Factor 1 alpha Is a Marker for an Unfavorable Prognosis in Early-Stage Invasive Cervical Cancer. Cancer Research. 60, 4693-4696 (2000).
- Harris, A. L. Hypoxia – a key regulatory factor in tumour growth. Nat. Rev. Cancer. 2, 38-47 (2002).
- Ramirez-Bergeron, D. L. Hypoxia affects mesoderm and enhances hemangioblast specification during early development. Development. 131, 4623-4634 (2004).
- Staff, F. E. Wheel-well Stowaways Risk Lethal Levels of Hypoxia and Hypothermia. Human Factors and Aviation. 44, 1-5 (1997).
- Shen, C., Powell-Coffman, J. A. Genetic Analysis of Hypoxia Signaling and Response in C. elegans. Annals of the New York Academy of Sciences. 995, 191-199 (2003).
- Shen, C., Nettleton, D., Jiang, M., Kim, S. K., Powell-Coffman, J. A. Roles of the HIF-1 Hypoxia-inducible Factor during Hypoxia Response in Caenorhabditis elegans. Journal of Biological Chemistry. 280, 20580-20588 (2005).
- Wang, G. L., Jiang, B. H., Rue, E. A., Semenza, G. L. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proceedings of the National Academy of Sciences. 92, 5510-5514 (1995).
- Epstein, A. C. R. C. elegans EGL-9 and Mammalian Homologs Define a Family of Dioxygenases that Regulate HIF by Prolyl Hydroxylation. Cell. 107, 43-54 (2001).
- Nystul, T. G., Goldmark, J. P., Padilla, P. A., Roth, M. B. Suspended Animation in C. elegans Requires the Spindle Checkpoint. Science. 302, 1038-1041 (2003).
- Gray, J. M. Oxygen sensation and social feeding mediated by a C. elegans guanylate cyclase homologue. Nature. 430, 317-322 (2004).
- Miller, D. L., Roth, M. B. C. Elegans Are Protected from Lethal Hypoxia by an Embryonic Diapause. Current Biology. 19, 1233-1237 (2009).
- Padilla, P. A., Nystul, T. G., Zager, R. A., Johnson, A. C. M., Roth, M. B. Dephosphorylation of Cell Cycle-regulated Proteins Correlates with Anoxia-induced Suspended Animation in Caenorhabditis elegans. Molecular Biology of the Cell. 13, 1473-1483 (2002).
- Hu, C. -. J., Wang, L. -. Y., Chodosh, L. A., Keith, B., Simon, M. C. Differential Roles of Hypoxia-Inducible Factor 1{alpha} (HIF-1{alpha}) and HIF-2{alpha} in Hypoxic Gene Regulation. Molecular and Cellular Biology. 23, 9361-9374 (2003).
- Treinin, M. HIF-1 is required for heat acclimation in the nematode Caenorhabditis elegans. Physiological Genomics. 14, 17-24 (2003).
- Miller, D. L., Roth, M. B. Hydrogen sulfide increases thermotolerance and lifespan in Caenorhabditis elegans. PNAS. 104, 20618-20622 (2007).
- Stiernagle, T. Maintenance of C. elegans. WormBook. , 1-11 (2006).
- Massie, M. R., Lapoczka, E. M., Boggs, K. D., Stine, K. E., White, G. E. Exposure to the metabolic inhibitor sodium azide induces stress protein expression and thermotolerance in the nematode Caenorhabditis elegans. Cell Stress Chaperones. 8, 1-7 (2003).
- Salceda, S., Caro, J. Hypoxia-inducible Factor 1α (HIF-1α) Protein Is Rapidly Degraded by the Ubiquitin-Proteasome System under Normoxic Conditions. Journal of Biological Chemistry. 272, 22642-22647 (1997).
- Theilacker, J. C., White, M. J. Diffusion of Gases in Air and Its Affect on Oxygen Deficiency Hazard Abatement. AIP Conference Proceedings. 823, 305-312 (2006).
- Chua, B., Kao, R. L., Rannels, D. E., Morgan, H. E. Inhibition of protein degradation by anoxia and ischemia in perfused rat hearts. Journal of Biological Chemistry. 254, 6617-6623 (1979).
- Probst, G., Riedinger, H., Martin, P., Engelcke, M., Probst, H. Fast Control of DNA Replication in Response to Hypoxia and to Inhibited Protein Synthesis in CCRF-CEM and HeLa Cells. Biological Chemistry. 380, 1371-1382 (1999).
- Semenza, G. L., Sen, C. K. . Oxygen Sensing. 381, (2004).
- Chan, K., Roth, M. B. Anoxia-Induced Suspended Animation in Budding Yeast as an Experimental Paradigm for Studying Oxygen-Regulated Gene Expression. Eukaryotic Cell. 7, 1795-1808 (2008).
- Nystul, T. G., Roth, M. B. Carbon monoxide-induced suspended animation protects against hypoxic damage in Caenorhabditis elegans. PNAS. 101, 9133-9136 (2004).
Cite This Article
Fawcett, E. M., Horsman, J. W., Miller, D. L. Creating Defined Gaseous Environments to Study the Effects of Hypoxia on C. elegans. J. Vis. Exp. (65), e4088, doi:10.3791/4088 (2012).