भेषज और कार्यात्मक जेनेटिक Assays Planarian की पुनर्जनन में हेरफेर करने के लिए बिही Dugesia</em
Summary
एक जीवित जानवर के भीतर स्टेम सेल भेदभाव के अध्ययन के लिए एक आकर्षक मॉडल planarian flatworm है. पुनर्जनन सरल विच्छेदन प्रयोगों है कि आसानी से एक बुनियादी प्रयोगशाला में प्रदर्शन कर रहे हैं के द्वारा अध्ययन किया है और (औषधीय और आनुवंशिक उत्तरदायी हैं<em> Vivo में</em> आरएनएआई) हेरफेर के रूप में इस अनुच्छेद में प्रोटोकॉल के द्वारा विस्तृत.
Abstract
Free-living planarian flatworms have a long history of experimental usage owing to their remarkable regenerative abilities1. Small fragments excised from these animals reform the original body plan following regeneration of missing body structures. For example if a ‘trunk’ fragment is cut from an intact worm, a new ‘head’ will regenerate anteriorly and a ‘tail’ will regenerate posteriorly restoring the original ‘head-to-tail’ polarity of body structures prior to amputation (Figure 1A).
Regeneration is driven by planarian stem cells, known as ‘neoblasts’ which differentiate into ~30 different cell types during normal body homeostasis and enforced tissue regeneration. This regenerative process is robust and easy to demonstrate. Owing to the dedication of several pioneering labs, many tools and functional genetic methods have now been optimized for this model system. Consequently, considerable recent progress has been made in understanding and manipulating the molecular events underpinning planarian developmental plasticity2-9.
The planarian model system will be of interest to a broad range of scientists. For neuroscientists, the model affords the opportunity to study the regeneration of an entire nervous system, rather than simply the regrowth/repair of single nerve cell process that typically are the focus of study in many established models. Planarians express a plethora of neurotransmitters10, represent an important system for studying evolution of the central nervous system11, 12 and have behavioral screening potential13, 14.
Regenerative outcomes are amenable to manipulation by pharmacological and genetic apparoaches. For example, drugs can be screened for effects on regeneration simply by placing body fragments in drug-containing solutions at different time points after amputation. The role of individual genes can be studied using knockdown methods (in vivo RNAi), which can be achieved either through cycles of microinjection or by feeding bacterially-expressed dsRNA constructs8, 9, 15. Both approaches can produce visually striking phenotypes at high penetrance- for example, regeneration of bipolar animals16-21. To facilitate adoption of this model and implementation of such methods, we showcase in this video article protocols for pharmacological and genetic assays (in vivo RNAi by feeding) using the planarian Dugesia japonica.
Protocol
Discussion
प्रोटोकॉल यहाँ planarian Dugesia के उत्थान बिही के लिए अध्ययन और छेड़खानी विस्तार assays का वर्णन. वे सरल कर रहे हैं और विशेष उपकरणों की आवश्यकता होती है जैसे कि वे आसानी से प्रयोगशाला या कक्षा में किया जा सकता नहीं है. Assays व्यक्तिगत प्रदर्शन किया जा सकता है है, या संयुक्त (vivo में दवा efficacies की रासायनिक आनुवंशिक स्क्रीनिंग के लिए) और उम्मीदवार जीन स्तर पर निष्पादित किया जा सकता है, या निष्पक्ष, उच्च throughput 8 स्क्रीनिंग के लिए अनुकूलनीय रहे हैं. चाहे अपने ही अधिकार में planarians की साज़िश जीव विज्ञान के अध्ययन के लिए, या ऊतक उत्थान के अध्ययन के लिए एक वैकल्पिक मॉडल में स्तनधारी homologues के vivo समारोह में आकलन करने के लिए, इन तरीकों के शोधकर्ताओं की एक विविध रेंज से ब्याज उत्प्रेरित करना चाहिए.
Declarações
The authors have nothing to disclose.
Acknowledgements
प्रयोगशाला में कार्य (MCB0919933) NSF और NIH (GM088790) द्वारा समर्थित है.
Materials
| Reagent | Vendor | Catalogue Number | Comments |
|---|---|---|---|
| Spring water | Kandiyohi. Premium Waters Inc. Minneapolis, MN | n/a | Other forms of spring water work well also. Trial first in viability assays. |
| 1 x buffered Montjuïch salts: NaCl (1.6mM), CaCl2 (1mM), MgSO4 (1mM), MgCl2 (0.1mM), KCl (0.1mM), NaHCO3 (1.2mM), HEPES (1.5mM). pH 7.4 at 24°C. | Multiple vendors | n/a | Artificial water for drug treatments during regenerative assays to ensure pH buffering. 5/8 Holtfreter’s solution is an alternative. |
| 2xYT Broth | Fisher Scientific | BP2467-500 | Media = 31 g/L . Autoclaved. |
| Petri Dish (100x25mm) | Fisher Scientific | 08-757-11 | Housing worms during regeneration cycles |
| Square Dish (100x100x15mm) | Fisher Scientific | 08-757-11A | Fill with water, freeze for ice tray used as worm cutting surface |
| Plastic tub: Ziploc Twist ‘n Loc (16oz). | Various retailers | n/a | Convenient water tight containers for RNAi cohorts |
| Chicken Liver | Commercial grocery | n/a | Bias towards organic supplies, owing to avoidance of antibiotics. |
| Hand Blender | Any kitchen supplier | n/a | Use for making chicken liver puree |
| Wire 1mm Mesh strainer | Any kitchen supplier | n/a | Use for straining chicken liver puree |
| Bovine red blood cells | Lampire Biological Laboratories | 7240807 | 100% Washed and pooled RBC suspension |
| Circular filter papers | Whatman #3 | 1003 055 | |
| Transfer Pipette | Fisher Scientific | 13-711-41 | |
| Sterile, surgical blades | Multiple Vendors | n/a | |
| ±Praziquantel | Sigma Aldrich | P4668 | Store powder aliquots in the dark at 4°C. Desiccate. |
| Dj-six-1 | GenBank AJ557022.1 | RNAi positive control for “eye-less” phenotype23 | |
| Dj-βcatenin-1 | GenBank AB181913.1 | RNAi positive control for two-headed phenotype17 | |
| Dj-PC2 | RNAi positive control for loss of light aversion phenotype24 |
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