आरएनएआई effectors के बैक्टीरियल डिलिवरी: Transkingdom आरएनएआई
Summary
शाही सेना हस्तक्षेप (आरएनएआई) आधारित चिकित्सा के विकास के लिए, एक उपन्यास की रणनीति विकसित की है, transkingdom आरएनएआई (tkRNAi). इस तकनीक गैर रोगजनक बैक्टीरिया का उपयोग करता है उत्पादन और लक्ष्य कोशिकाओं में चिकित्सीय कम बाल के लिये कांटा शाही सेना (shRNA) देने. यहाँ, tkRNAi शास्त्रीय ABCB1 की मध्यस्थता कैंसर कोशिकाओं के बहुऔषध प्रतिरोध (एमडीआर) के उत्क्रमण के लिए सफलतापूर्वक लागू किया गया था.
Abstract
RNA interference (RNAi) represents a high effective mechanism for specific inhibition of mRNA expression. Besides its potential as a powerful laboratory tool, the RNAi pathway appears to be promising for therapeutic utilization. For development of RNA interference (RNAi)-based therapies, delivery of RNAi-mediating agents to target cells is one of the major obstacles. A novel strategy to overcome this hurdle is transkingdom RNAi (tkRNAi). This technology uses non-pathogenic bacteria, e.g. Escherichia coli, to produce and deliver therapeutic short hairpin RNA (shRNA) into target cells to induce RNAi. A first-generation tkRNAi-mediating vector, TRIP, contains the bacteriophage T7 promoter for expression regulation of a therapeutic shRNA of interest. Furthermore, TRIP has the Inv locus from Yersinia pseudotuberculosis that encodes invasin, which permits natural noninvasive bacteria to enter β1-integrin-positive mammalian cells and the HlyA gene from Listeria monocytogenes, which produces listeriolysin O. This enzyme allows the therapeutic shRNA to escape from entry vesicles within the cytoplasm of the target cell. TRIP constructs are introduced into a competent non-pathogenic Escherichia coli strain, which encodes T7 RNA polymerase necessary for the T7 promoter-driven synthesis of shRNAs. A well-characterized cancer-associated target molecule for different RNAi strategies is ABCB1 (MDR1/P-glycoprotein, MDR1/P-gp). This ABC-transporter acts as a drug extrusion pump and mediates the “classical” ABCB1-mediated multidrug resistance (MDR) phenotype of human cancer cells which is characterized by a specific cross resistance pattern. Different ABCB1-expressing MDR cancer cells were treated with anti-ABCB1 shRNA expression vector bearing E. coli. This procedure resulted in activation of the RNAi pathways within the cancer cells and a considerable down regulation of the ABCB1 encoding mRNA as well as the corresponding drug extrusion pump. Accordingly, drug accumulation was enhanced in the pristine drug-resistant cancer cells and the MDR phenotype was reversed. By means of this model the data provide the proof-of-concept that tkRNAi is suitable for modulation of cancer-associated factors, e.g. ABCB1, in human cancer cells.
Protocol
Discussion
सेल नंबर के संक्रमण के लिए वरीयता प्राप्त और इसी MOI इस्तेमाल किया, गंभीर अवलोकन के अंतर्गत सेल लाइनों और विकास की अपनी गति पर निर्भर है. बोने के लिए इष्टतम सेल नंबर मिल करने के लिए, विकास की गति निर्धारित ?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
कार्य कोई अनुदान द्वारा समर्थित किया गया. 01GU0615 "Bundesministerium फर Forschung und प्रौद्योगिकियों (BMBF).
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Agar | Biochrom AG | 214050 | ||
| Amphotericin B | Biochrom AG | A2612 | ||
| Dulbecco’s Phosphate Buffered Saline-PBS (10 x) | Invitrogen GmbH | 14080048 | ||
| E. coli ceq221 | Cequent Pharmaceuticals Inc. | No catalogue available, direct order | ||
| Gentamycin | Biochrom AG | A2710 | ||
| Penicillin/Streptomycin | Invitrogen GmbH | Contains 5,000 units of penicillin (base) and 5,000 μg of streptomycin (base)/ml utilizing penicillin G (sodium salt) and streptomycin sulfate in 0.85% saline. | ||
| Sodium chloride | Merck KgaA | 1064060500 | ||
| Tryptone | Difco Laboratories | 211705 | ||
| Yeast Extract | Difco Laboratories | 212750 |
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