Zebrafish लार्वा में गैर इनवेसिव कैंडिडिआसिस फैलाया इमेजिंग
Summary
तेजी से विकास, छोटे आकार और zebrafish की पारदर्शिता सहज प्रतिरक्षा संक्रमण के नियंत्रण के अध्ययन के लिए काफी लाभ कर रहे हैं<sup1-4></sup>. यहाँ हम zebrafish लार्वा संक्रमण कवक रोगज़नक़ का उपयोग करने के लिए तकनीक का प्रदर्शन<em> Candida albicans</emMicroinjection> पद्धति हाल ही में के कवक द्विरूपता के नियंत्रण में भक्षककोशिकीय NADPH oxidase गतिविधि फंसाना किया<sup> 5</sup>.
Abstract
Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host.
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color.
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.
Protocol
Discussion
zebrafish microinjection यहाँ प्रस्तुत विधि Gutzman एट अल से अलग 34. कि यहाँ में हम 36-48 HPF लार्वा के hindbrain निलय में कान का पुटिका के माध्यम से इंजेक्शन दिखाना है. विधि का वर्णन हम कम ऊतकों को नुकसान के साथ hindbrain निलय में 10-15 ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
लेखकों के लिए microinjection, सलाह के लिए प्रशिक्षण Clarissa हेनरी के लिए भ्रूण और उपकरणों के विकास का उपयोग तेजी पर डॉ. कैरोल किम की प्रयोगशाला का शुक्रिया अदा करना, और fli1 योगदान के लिए नाथन लॉसन: EGFP मछली. हम पांडुलिपि के महत्वपूर्ण पढ़ने के लिए व्हीलर प्रयोगशाला और शॉन दीवारों के सदस्यों को धन्यवाद. हम भी इस परियोजना पर तकनीकी सलाह के लिए मछली देखभाल और सलाह के लिए मार्क, और Nilan रयान Phennicie और क्रिस्टिन गेबर धन्यवाद देना चाहूंगा. इस काम के. ब्रदर्स, MAFES हैच E08913-08 अनुदान, और एनआईएच NCRR आर व्हीलर P20RR016463 पुरस्कार के लिए एक MAFES अनुसंधान सहायता द्वारा वित्त पोषित किया गया था.
Materials
| Name of the reagent | Company | Catalog number | Comments (optional) |
| Spawning tanks | Aquatic habitats | 2L | |
| 1.7 mL tubes | Axygen | MCT-175-C | |
| Instant Ocean | Fisher Scientific | S17957C | |
| Extra deep Petri dishes | Fisher Scientific | 08-757-11Z | |
| Standard Petri dishes | VWR Scientific | 25384-302 | |
| Transfer pipettes | Fisher Scientific | 13-711-7M | |
| Yeast Extract | VWR Scientific | 90000-726 | |
| Peptone | VWR Scientific | 90000-264 | |
| Dextrose | Fisher Scientific | D16-1 | |
| Agar | VWR Scientific | 90000-760 | |
| Disposable Hemocytometer | VWR Scientific | 82030-468 | |
| Phosphate Buffered Saline | VWR Scientific | 12001-986 | |
| Dumont Dumoxel Tweezers | VWR Scientific | 100501-806 | |
| Wooden Dowels | VWR Scientific | 10805-018 | |
| KimWipes | VWR Scientific | 300053-964 | |
| Low Melt Agarose | VWR Scientific | 12001-722 | |
| Agarose for injection dishes | VWR Scientific | 12002-102 | |
| Flaming Brown Micropipette Puller | Sutter Instruments | P-97 | |
| Hollow glass rods | Sutter Instruments | BF120-69-10 | For glass rods smooth glass by heating over bunsen burner |
| Pipette Storage Box | Sutter Instruments | BX10 | |
| MPPI-3 Injection system | Applied Scientific Instrumentation | MPPI-3 | |
| Back Pressure Unit | Applied Scientific Instrumentation | BPU | |
| Micropipette Holder kit | Applied Scientific Instrumentation | MPIP | |
| Foot Switch | Applied Scientific Instrumentation | FSW | |
| Micromanipulator | Applied Scientific Instrumentation | MM33 | |
| Magnetic Base | Applied Scientific Instrumentation | Magnetic Base | |
| Tricaine methane sulfonate | Western Chemical Inc. | MS-222 | |
| Dissecting Scope | Olympus | SZ61 top SZX-ILLB2-100 base | |
| Confocal Microscope | Olympus | IX-81 with FV-1000 laser scanning confocal system | |
| TC-7 Tissue Culture Roller drum with 14 inch test tube wheel | New Brunswick Scientific | TC-7 | |
| Imaging Dishes | MatTek Corporation | P24G-1.0-10-F | |
| Pipette tips for loading needles | Eppendorf | 930001007 | |
| Plate pouring grids | Adaptive Science Tools | TU-1 | |
| Heated Stage | Bioptechs Inc. | Delta T-5 | |
| Flat Spatula | VWR Scientific | 82027-486 | |
| Plastic Sieves | Wares of Knutsford Online | 12 cm | |
| Parafilm | VWR Scientific | 52858-000 | |
| Vortex Genie | VWR Scientific | 14216-184 | |
| 16 x 150 mm Culture tubes | VWR Scientific | 60825-435 | |
| Nanodrop | Thermo Scientific | ND 2000 | |
| Phenol Red | VWR Scientific | 97062-478 | |
| HCl | VWR Scientific | 87003-216 | |
| NaCl | VWR Scientific | BDH4534-500GP | |
| KCl | VWR Scientific | BDH4532-500GP | |
| MgSO4 | VWR Scientific | BDH0246-500GP | |
| Ca(NO3)2 | VWR Scientific | BDH0226-500GP | |
| HEPES | VWR Scientific | BDH4520-500GP | |
| Morpholinos | GeneTools, LLC |
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