高效转染树突状细胞的无细胞的成熟优化的协议
Summary
我们目前我们优化的高通量nucleofection协议，要么质粒DNA或不会造成细胞的成熟的siRNA转染初级人类单核细胞衍生的树突状细胞的有效方法。我们进一步提供证据证明为成功的siRNA沉默靶基因的mRNA和蛋白水平的RIG – I。
Abstract
树突状细胞可以被认为是其中发挥关键作用，在其发起和响应感染 1的免疫系统的哨兵。天真区议会致病抗原的检测是通过模式识别受体（PRRS）是能够识别特定病原体相关分子模式（PAMPS）的保守结构。区议会PAMPS检测触发细胞内的信号级联，导致他们的活化和转化为成熟DC。这个过程通常是通过对1型干扰素的生产特点以及与其他炎性细胞因子，如MHCII和CD86和迁移的淋巴结，与T细胞相互作用启动适应性免疫反应的成熟DC的细胞表面标志的上调， 3。因此，区议会的联系先天免疫和适应性免疫系统。
解剖的基本直流响应，以各种病原体的分子网络的能力是至关重要的，以更好地了解了这些信号通路的调控和诱导基因。它也应当有助于促进DC为基础的疫苗针对传染病和肿瘤的发展。然而，此行的研究已经严重阻碍，难以转染的主要区议会 4 。
病毒转导的方法，如慢病毒系统，通常使用的，但携带很多限制，如复杂性和有害生物风险（ 相关费用）5,6,7,8。此外，该病毒基因产物的交付增加转区议会 9,10,11,12的免疫原性。电穿孔技术已用于13,14,15结果好坏参半，但我们是第一个报告使用一种高通量的转染协议和确凿证明其效用。
在这份报告中，我们总结了一个优化的商业协议，高通量转染人类的主要区议会，与有限的细胞毒性和一个 DC成熟16的情况下。转染效率（GFP质粒）和细胞活力分别超过50％和70％。流式细胞仪分析建立的成熟标志物CD86和MHCII转染细胞中表达增加的情况下，而定量RT – PCR结果显示没有IFNβ上调。使用此电协议，我们提供证据议会成功转染siRNA和有效的敲有针对性的基因RIG – I，16,17一个关键的病毒识别受体的mRNA和蛋白水平，。
Protocol
1。计划Amaxa 96穿梭Nucleofector 打开一个新的参数文件。 选择您将使用标准的转拖动光标在96孔板图，井的数量。使用至少3口井，池，每个实验样品。 输入程序代码：在第一部分选择“FF”，并在第2部分选择'168'从下拉式菜单从解决方案中，选择“单核细胞，人类” 在控制选项，选择“标准”。 点击应用。 包括无染控制，从图中选择进一步根?…
Discussion
幼稚的初级树突状细胞的高效转染重要的是，在这关键的细胞介导的先天适应性免疫过渡的细胞炎症通路的高通量分析和逆向工程。然而，大多数研究人员发现，这些细胞是难以转染效率和转染过程诱导细胞成熟时，使用标准的转染技术。我们研究了克服这些限制是否可以由高吞吐量协议优化使用，同时，独立96以及商业核转染系统（龙沙）。
利用荧光激活细胞分选（FAC…
Declarações
The authors have nothing to disclose.
Acknowledgements
该项目是由美国国立卫生研究院NIAID的合同号HHSN2662000500021C支持。我们感谢他的技术援助名臣。
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 |
Referências
- Reis e Sousa, C. Activation of dendritic cells: translating innate into adaptive immunity. Curr. Opin. Immunol. 16, 21-25 (2004).
- Bancherau, J., Steinman, R. M. Dendritic cells and the control of immunity. Nature. 392, 245-252 (1998).
- Clark, G. J. The role of dendritic cells in the innate immune system. Microbes Infect. 2, 257-272 (2000).
- Hamm, A. Efficient transfection method for primary cells. Tissue Eng. 8, 235-245 (2002).
- Henderson, R. A. Human dendritic cells genetically engineered to express high levels of the human epithelial tumor antigen mucin (MUC-1). Cancer Res. 56, 3763-3770 (1996).
- Reeves, M. E. Retroviral transduction of human dendritic cells with a tumor-associated antigen gene. Cancer Res. 56, 5672-5677 (1996).
- Aicher, . Successful retroviral mediated transduction of a reporter gene in human dendritic cells: feasibility of therapy with gene-modified antigen presenting cells. Exp. Hematol. 25, 39-44 (1997).
- Thomas, C. E. Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet. 4, 346-358 (2003).
- Jooss, K. Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle fibers. J. Virol. 72, 4212-4223 (1998).
- Mitchell, D. A. RNA-transfected dendritic cells in cancer immunotherapy. J. Clin. Invest. 106, 1065-1069 (2000).
- Mincheff, M. In vivo transfection and/or cross-priming of dendritic cells following DNA and adenoviral immunizations for immunotherapy of cancer-changes in peripheral mononuclear subsets and intracellular IL-4 and IFN-gamma lymphokine profile. Crit. Rev. Oncol. Hematol. 39, 125-132 (2001).
- Roth, . Helper-dependent adenoviral vectors efficiently express transgenes in human dendritic cells but still stimulate antiviral immune responses. J. Immunol. 169, 4651-4656 (2002).
- Tendeloo, V. F. V. a. n. Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. Blood. 98, 49-56 (2001).
- Lenz, P. Nucleoporation of dendritic cells: efficient gene transfer by electroporation into human monocyte-derived dendritic cells. FEBS Lett. 538, 149-154 (2003).
- Prechtel, A. T. Small interfering RNA (siRNA) delivery into monocyte-derived dendritic cells by electroporation. J. Immunol. Methods. 311, 139-152 (2006).
- Bowles, R. Validation of efficient high-throughput plasmid and siRNA transfection of human monocyte-derived dendritic cells without cell maturation. J. Immunol. Methods. , .
- Kato, H. Cell type-specific involvement of RIG-I in antiviral response. Immunity. 23, 19-28 (2005).
- Kato, H. Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature. 441, 101-105 (2006).
- Haller, O. The Mx GTPase family of interferon-induced antiviral proteins. Microbes Infect. 9, 1636-1643 (2007).
Tags
Dendritic CellsImmune SystemInfectionPathogen-associated Molecular Patterns (PAMPs)Pattern Recognition Receptors (PRRs)Cell MaturationType 1 InterferonProinflammatory CytokinesMHCIICD86Adaptive Immune ResponseMolecular NetworksSignaling PathwaysInduced GenesDC-based VaccinesTransfection Of Dendritic Cells
Citar este artigo
Bowles, R., Patil, S., Pincas, H., Sealfon, S. C. Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation. J. Vis. Exp. (53), e2766, doi:10.3791/2766 (2011).