هذه تفاصيل ورقة كيفية استخدام الدفق المستمر غرف نقص الأكسجين لتوليد أجواء بتركيزات محددة من O<sub> 2</sub> لفهم الاستجابات البيولوجية إلى انخفاض O<sub> 2</sub>. هذا النظام هو سهل لإعداد وصيانة، ومرنة بما فيه الكفاية لتلائم مجموعة واسعة من O<sub> 2</sub> تجمعات ونظم نموذج
Oxygen is essential for all metazoans to survive, with one known exception1. Decreased O2 availability (hypoxia) can arise during states of disease, normal development or changes in environmental conditions2-5. Understanding the cellular signaling pathways that are involved in the response to hypoxia could provide new insight into treatment strategies for diverse human pathologies, from stroke to cancer. This goal has been impeded, at least in part, by technical difficulties associated with controlled hypoxic exposure in genetically amenable model organisms.
The nematode Caenorhabditis elegans is ideally suited as a model organism for the study of hypoxic response, as it is easy to culture and genetically manipulate. Moreover, it is possible to study cellular responses to specific hypoxic O2 concentrations without confounding effects since C. elegans obtain O2 (and other gasses) by diffusion, as opposed to a facilitated respiratory system6. Factors known to be involved in the response to hypoxia are conserved in C. elegans. The actual response to hypoxia depends on the specific concentration of O2 that is available. In C. elegans, exposure to moderate hypoxia elicits a transcriptional response mediated largely by hif-1, the highly-conserved hypoxia-inducible transcription factor6-9. C .elegans embryos require hif-1 to survive in 5,000-20,000 ppm O27,10. Hypoxia is a general term for “less than normal O2“. Normoxia (normal O2) can also be difficult to define. We generally consider room air, which is 210,000 ppm O2 to be normoxia. However, it has been shown that C. elegans has a behavioral preference for O2 concentrations from 5-12% (50,000-120,000 ppm O2)11. In larvae and adults, hif-1 acts to prevent hypoxia-induced diapause in 5,000 ppm O212. However, hif-1 does not play a role in the response to lower concentrations of O2 (anoxia, operational definition <10 ppm O2)13. In anoxia, C. elegans enters into a reversible state of suspended animation in which all microscopically observable activity ceases10. The fact that different physiological responses occur in different conditions highlights the importance of having experimental control over the hypoxic concentration of O2.
Here, we present a method for the construction and implementation of environmental chambers that produce reliable and reproducible hypoxic conditions with defined concentrations of O2. The continual flow method ensures rapid equilibration of the chamber and increases the stability of the system. Additionally, the transparency and accessibility of the chambers allow for direct visualization of animals being exposed to hypoxia. We further demonstrate an effective method of harvesting C. elegans samples rapidly after exposure to hypoxia, which is necessary to observe many of the rapidly-reversed changes that occur in hypoxia10,14. This method provides a basic foundation that can be easily modified for individual laboratory needs, including different model systems and a variety of gasses.
هذا الأسلوب يعرض استراتيجية لبناء بيئة نقص الأوكسجين الذي يسمح للبيئات مع تركيز دقيق من الأوكسجين إلى الحفاظ عليها في المختبر. هذه الغرف توفر طريقة بسيطة لكشف الكائنات الحية إلى تركيزات منخفضة محددة من O 2 ورصد مخرجات الجزيئية والفيزيولوجية. يتم تجميعها في غرف?…
The authors have nothing to disclose.
ونحن نشكر أعضاء مختبر ميلر للمناقشة وقراءة نقدية للمخطوطة. وأيد هذا العمل من قبل على جائزة محقق جديد من مركز الصدمة ناثان من التميز في علم الأحياء الأساسي للشيخوخة إلى DLM والمعاهد الوطنية للصحة جائزة R00 AG030550 إلى DLM.
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 |