Planarian स्थिरीकरण, आंशिक किरणन, और ऊतक प्रत्यारोपण
Summary
Planaria के बीच परिभाषित और लगातार आकार के ऊतक की कलम बांधने का काम के लिए एक प्रभावी तरीका वर्णित है. भी शामिल कैसे स्थिरीकरण प्रत्यारोपण के लिए इस्तेमाल तकनीक जीवित पशुओं का आंशिक विकिरण के लिए नेतृत्व ढाल, के साथ संयोजन के रूप में अनुकूलित कर सकते हैं की एक विवरण.
Abstract
The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts3. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal4, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.
Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a naïve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.
Both tissue transplantation5,6 and partial irradiation7 have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of naïve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals8. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.
Protocol
Discussion
स्थिरीकरण का महत्व
स्थिरीकरण द्वारा अब तक इन प्रक्रियाओं में से किसी के सफल समापन के लिए सबसे महत्वपूर्ण कदम है. यदि planaria अनुचित तरीके से आंशिक विकिरण से पहले स्थिर रहे हैं, वे नेतृत्व ढाल के न?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
लेखकों के लिए planarian प्रत्यारोपण पर उपयोगी सलाह के रूप में के रूप में अच्छी तरह से सांचेज़ प्रयोगशाला के अतीत और वर्तमान सदस्यों के लिए इन तकनीकों के विकास के दौरान अमूल्य विचार विमर्श के लिए Chiyoko कोबायाशी और Kiyokazu Agata धन्यवाद करना चाहते हैं. यह काम एनआईएच प्रशिक्षण OCG और एएसए के लिए एनआईएच R37GM057260 अनुदान (HD0791 5T32) द्वारा समर्थित किया गया था. एएसए एक हॉवर्ड ह्यूजेस मेडिकल इंस्टीट्यूट अन्वेषक है.
Materials
| Name of the reagent | Company | Catalogue number |
| General Purpose Transfer Pipette | Samco | 691 |
| Capillary tubes (ID 0.75 mm) | FHC | 30-30-0 |
| Capillary tubes (OD 0.7 mm) | FHC | 30-50-08 |
| Parafilm M | VWR | 52858-076 |
| Kimwipes 34155 | VWR | 500029-891 |
| Black filter paper | Schleicher & Schuell | 10310809 |
| Whatman #2 filter paper 1002-055 | Fisher Scientific | 09-810B |
| Whatman #3 filter paper 1003-185 | Fisher Scientific | 09-820E |
| Cigarette rolling paper | Zig-Zag, original | NA |
| Petri dishes | VWR | 82050-544 |
| Forceps DUMONT, INOX #5 | FST | 11251-20 |
| Chloretone | Sigma Aldrich | 112054 |
| Casein | Sigma Aldrich | C3400 |
| Lead Shields | Alpha Systems Corp., Bluffdale, UT | Custom design |
| XRAD-320 Biological Irradiator | Precision X-Ray, North Branford, CT | NA |
Riferimenti
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