आण्विक प्रतिरोपित स्नायु पूर्वपुस्र्ष कोशिकाओं को लक्षित इमेजिंग
Summary
एक गैर इनवेसिव myoblast प्रत्यारोपण की सफलता का मूल्यांकन करने का मतलब वर्णित है. विधि एक एकीकृत संलयन रिपोर्टर जीन जीन जिनकी अभिव्यक्ति विभिन्न इमेजिंग रूपात्मकता के साथ imaged किया जा सकता है की रचना का लाभ लेता है. यहाँ, हम एक का इस्तेमाल करते हैं<em> Fluc</em> रिपोर्टर जीन अनुक्रम bioluminescence इमेजिंग के माध्यम से कोशिकाओं को लक्षित है.
Abstract
Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.
Protocol
Representative Results
Discussion
इस अध्ययन में, हम प्रत्यारोपण के बाद एक तेज और विश्वसनीय आणविक इमेजिंग, रिपोर्टर जीन दृष्टिकोण गैर invasively लक्ष्य myoblasts / MPCs का वर्णन किया है. जबकि इस अध्ययन के माध्यम से bioluminescense (BLI) इमेजिंग, जिस तरीके में कोशि?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
लेखकों के लिए संजीव गंभीर fluc/mrfp/sr39tk रिपोर्टर जीन के उपहार के लिए धन्यवाद देना चाहूंगा. इस काम कनाडा के स्टेम सेल (SCN) नेटवर्क, और यिशै यात्रा फाउंडेशन द्वारा वित्त पोषित किया गया था.
Materials
| Name of reagent | Company | Catalogue number | Comments (optional) |
| C2C12 myoblasts | ATCC | ||
| Dulbecco’s Modified Eagle’s Medium | Life Technologies | 12800-017 | |
| fetal bovine serum | Life Technologies | 12483020 | |
| 0.25% Trypsin-EDTA | Life Technologies | 25200-72 | |
| Hanks Balanced Salt Solution | Sigma Aldrich | H6648 | |
| Lipofectamine 2000 | Life Technologies | 11668-019 | |
| Nikon Eclipse TE2000-5 | Nikon Instruments Inc. | ||
| Xenolight D-luciferin | PerkinElmer | 122796 | 40 mg/ml in PBS |
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