सेल परिपक्वता के बिना वृक्ष के समान कोशिकाओं के कुशल अभिकर्मक के लिए अनुकूलित प्रोटोकॉल
Summary
हम या तो प्लाज्मिड या सेल परिपक्वता के कारण के बिना डीएनए siRNA साथ प्राथमिक मानव monocyte व्युत्पन्न वृक्ष के समान कोशिकाओं transfecting की एक कारगर तरीका के रूप में हमारे अनुकूलित nucleofection उच्च throughput प्रोटोकॉल उपस्थित थे. हम आगे लक्षित जीन RIG मैं दोनों mRNA और प्रोटीन के स्तर पर सफल siRNA मुंह बंद करने के लिए सबूत प्रदान करते हैं.
Abstract
Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection of pathogenic antigen by naïve DCs is through pattern recognition receptors (PRRs) which are able to recognize specific conserved structures referred to as pathogen-associated molecular patterns (PAMPS). Detection of PAMPs by DCs triggers an intracellular signaling cascade resulting in their activation and transformation to mature DCs. This process is typically characterized by production of type 1 interferon along with other proinflammatory cytokines, upregulation of cell surface markers such as MHCII and CD86 and migration of the mature DC to draining lymph nodes, where interaction with T cells initiates the adaptive immune response2,3. Thus, DCs link the innate and adaptive immune systems.
The ability to dissect the molecular networks underlying DC response to various pathogens is crucial to a better understanding of the regulation of these signaling pathways and their induced genes. It should also help facilitate the development of DC-based vaccines against infectious diseases and tumors. However, this line of research has been severely impeded by the difficulty of transfecting primary DCs4.
Virus transduction methods, such as the lentiviral system, are typically used, but carry many limitations such as complexity and bio-hazardous risk (with the associated costs)5,6,7,8. Additionally, the delivery of viral gene products increases the immunogenicity of those transduced DCs9,10,11,12. Electroporation has been used with mixed results13,14,15, but we are the first to report the use of a high-throughput transfection protocol and conclusively demonstrate its utility.
In this report we summarize an optimized commercial protocol for high-throughput transfection of human primary DCs, with limited cell toxicity and an absence of DC maturation16. Transfection efficiency (of GFP plasmid) and cell viability were more than 50% and 70% respectively. FACS analysis established the absence of increase in expression of the maturation markers CD86 and MHCII in transfected cells, while qRT-PCR demonstrated no upregulation of IFNβ. Using this electroporation protocol, we provide evidence for successful transfection of DCs with siRNA and effective knock down of targeted gene RIG-I, a key viral recognition receptor16,17, at both the mRNA and protein levels.
Protocol
Discussion
भोले प्राथमिक वृक्ष के समान कोशिकाओं के कुशल अभिकर्मक उच्च throughput विश्लेषण और इस कुंजी सहज अनुकूली प्रतिरक्षा संक्रमण mediating कक्ष में सेलुलर भड़काऊ रास्ते के रिवर्स इंजीनियरिंग के लिए महत्वपूर्ण है. हाल?…
Declarações
The authors have nothing to disclose.
Acknowledgements
परियोजना NIH NIAID अनुबंध सं. HHSN2662000500021C द्वारा समर्थित किया गया. हम अपने तकनीकी सहायता के लिए मिंग चेन धन्यवाद.
Materials
| Equipment/Reagent | Company | Catalogue # | Comments |
|---|---|---|---|
| Amaxa Nucleofector 96-well Shuttle | Lonza | 108S0109 | Serial number |
| Amaxa Human Monocyte 96-well Nucleofector Kit | Lonza | VHPA-2007 | Contains the Human Monocyte 96-well Nucleofector Solution, the 96-well Supplement and the Nucleocuvettes and plates |
| RIG-I siRNA | Dharmacon | L-012511-00 | |
| GLO siRNA | Dharmacon | D-001600-01-20 | |
| RPMI 1640 | Invitrogen | 11875 | Supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin to make DC growth medium |
| DMEM | Invitrogen | 11965 | |
| L-glutamine | Invitrogen | 25030081 | |
| Penicillin/streptomycin | Invitrogen | 15070063 | |
| Fetal Calf Serum | HyClone | 3070.03 | |
| Dendritic Cells | New York Blood center | DCs are purified from buffy coats using a standard procedure |
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