小RNA转染原代人Th17细胞通过下一代电穿孔
Summary
Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.
Abstract
CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from naïve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1β, IL-6, IL-23, and TGFβ. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, RORγt, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.
Introduction
CD4 + T细胞是适应性免疫应答的关键协调器。幼稚CD4 + T细胞是能够发育成几个不同的效应T细胞（ 例如 TH1，TH2，Th17细胞， 等 ），每个具有自己的一套特性的细胞因子和转录因子，取决于局部微环境1。血统决定T细胞使对于两个保护性免疫和容忍，自我批评。 Th17细胞是公知的，以打击胞外细菌和真菌的T细胞的一个子集，但它们的不当响应也牵涉在多种自身免疫疾病和炎性疾病的发病机理，例如多发性硬化和牛皮癣2,3。人Th17细胞可以从幼稚的CD4 + T细胞在体外通过提供一个适当的偏振环境4来生成。 VARIO我们的细胞因子的组合IL-1β，IL-23，TGFβ，和IL-6已被用于人Th17细胞的发展。人Th17细胞表达CCR6，即通常用于识别该细胞群，并通过它们的主要转录因子，RORγt（由RORC编码）5,6的表达被定义趋化因子受体。 Th17细胞具有表达多种细胞因子的能力，但IL-17A是由这些细胞产生的细胞谱系限定的效应细胞因子。我们检查了所有三个Th17细胞相关标记（CCR6，RORγt，IL-17A）的表达来评估我们的人Th17 体外分化检测的鲁棒性。此外，我们培养非偏振的条件下，在没有细胞因子或阻断抗体添加到培养基中作为阴性对照使用，因为这些Th17细胞标志物的表达应该是非常低的或不存在下的人CD4 + T细胞。
ve_content“>学习正常的人T细胞的发育和生物学的一种方法是其发展过程中操纵基因表达。短干扰RNA（siRNA）是靶向蛋白质编码的mRNA，并且可以被利用来降低特定基因表达的合成的小RNA分子。微RNA（miRNA）是公知的调节基因表达的转录后的内源性非编码小RNA。的miRNA已显示出在小鼠和人的T细胞生物学中起重要作用，包括在Th17细胞7，8，9，它关键的是要具有在人T细胞操纵的小RNA的活性，研究其对基因表达，并最终对人T细胞生物学效应的可靠的方法。在此，我们描述了我们用于引入开发了一种易于使用的，一致的和可靠的协议小合成的RNA和锁定核酸（LNA，化学修饰的核酸具有增加的稳定性）成免疫细胞，并且具体到人Th17细胞。有引入的小RNA到哺乳动物细胞中，其通常落入化学，生物或物理类别10的若干替代方法。常用的化学方法，包括基于脂质的转染和磷酸钙转染，依靠创建被细胞更有效地吸收化学-DNA复合物。一般而言，化学方法是不适合的原代T细胞的转染效率高。最常见的生物的方法是使用病毒载体（ 例如逆转录病毒或慢病毒），其直接插入外来RNA引入宿主作为其天然复制周期的一部分。病毒转导通常需要更长的时间来完成，特别是当一个在时间因素的原病毒的质粒分子克隆。此外，病毒转导载体可以是可能危害人体研究。电穿孔是一种物理米通过使细胞对高的电压脉冲，从而允许核酸以瞬时进入细胞在那里他们可以在他们的目标作用诱导膜通透性ethod。传统的电文书不能有效转染主要淋巴细胞。然而，优化的下一代电穿孔已经被证明是能够以很高的效率转染的T细胞，特别是当待转染的材料为小RNA。术语下一代是松散用来区分传统的电穿孔机两个新的平台（ 例如 ，霓虹灯，Amaxa公司）。此外，此方法是一种用于具有高达在单一实验中约120的小RNA，其通常使用验证合成试剂中等通量筛选容易扩展。重要的是，转染的成功可以在T细胞活化后只需16小时来实现。这种方法的缺点，但是，它不会导致基因组稳定incorporati上，因此是短暂的。因此，这是值得的额外的努力以创建可以被包装到病毒载体中并在T细胞在需要小RNA的长期表达的情况下成功地表达了稳定的表达构建体。
我们已经使用了下一代的转染（ 例如 ，氖），实现多样化合成的单链或双链RNA或LNA寡核苷酸的工具用于不同的目的11，12，13。有效的RNA干扰可以在原代小鼠和人T细胞使用双链短干扰RNA（siRNA）所诱导。这个协议描述了用于在人Th17细胞使用这种技术的优化条件。除了的siRNA，可商购的合成的miRNA模拟物和抑制剂可用于研究的miRNA增益和功能丧失。 miRNA模拟物是双链RNA分子非常相似的siRNA，但designe20d与内源成熟miRNA的序列。 miRNA的抑制剂是经化学修饰的结合天然miRNA和拮抗它们的功能的RNA和/或LNA基于单链寡核苷酸。我们发现，所有这些工具都可以在原代培养的T淋巴细胞有效地使用，包括但不限于人Th17细胞。
Protocol
Representative Results
Discussion
该协议提供用于递送的小RNA的成人Th17细胞的改进的方法。虽然人Th17细胞在此使用，电穿孔的这种方法与小RNA可以与其他主人T辅助细胞亚组，如TH1，TH2，以及调节性T细胞可以使用。它不是幼稚的CD4 + T细胞的运作良好，因此细胞必须培养的转染之前被激活。对于此协议中，我们首先优化了体外培养体系为更好的IL-17A的生产。最大的因素是基础的媒体。我们发现，无血清培养基优于传…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the US National Institutes of Health grants (R01HL109102, P01HL107202, U19CA179512, F31HL131361), a Leukemia & Lymphoma Society scholar award (K.M.A.), and the National Institute of General Medical Sciences (NIGMS) Medical Scientist Training Program (Grant #T32GM007618) (M.M.).
Materials
| anti-human IL-17A PE | ebioscience | 12-7179-42 | Clone: eBio64DEC17 |
| anti-human IFNg FITC | ebioscience | 11-7319-82 | Clone: 4S.B3 |
| anti-human CD4 eVolve605 | ebioscience | 83-0047-42 | Clone: SK3 |
| mouse anti-human CD196 (CCR6) BV421 | BD biosciences | 562515 | Clone: 11A9 |
| anti-human RORgt AF647 | BD biosciences | 563620 | Clone: Q21-559 |
| anti-human CD45 eFluor450 | ebioscience | 48-9459-42 | Clone: 2D1 |
| Foxp3/Trascription Factor Staining Buffer Set | ebioscience | 00-5523-00 | For intracellular transcription factor flow cytometry staining |
| Hu FcR Binding Inhibitor Purified | ebioscience | 14-9161-71 | |
| ImmunoCult-XF T Cell Expansion Medium | Stemcell Technologies | 10981 | "Serum-Free Base Media" |
| MACS CD28 pure functional grade, human | Miltenyi Biotec | 130-093-375 | Clone: 15E8 |
| anti-human CD3 Purified | UCSF monoclonal antibody core | N/A | Clone: OKT-3 |
| LEAF purified anti-human IL-4 | Biolegend | 500815 | Clone: MP4-25D2 |
| anti-human IFNg, functional grade purified | ebioscience | 16-7318-85 | Clone: NIB42 |
| Recombinant human IL-23 | Peprotech | 200-23 | |
| Recombinant human IL-1β | Peprotech | 200-01B | |
| Recombinant human TGF-β1 | Peprotech | 100-21C | |
| Recombinant human IL-6 | Peprotech | 200-06 | |
| siGENOME Control Pool, Non-targeting #2 | Dharmacon | D-001206-14-05 | |
| siGENOME SMARTpool Human RORC | Dharmacon | M-003442-00 | |
| siGENOME SMARTpool Human PTPRC | Dharmacon | M-008067-01 | |
| Dynabeads Untouched Human CD4 T Cells Kit | ThermoFisher Scientific | 11346D | Human CD4+ T cell Isolation Kit |
| Neon Tranfection System | ThermoFisher Scientific | MPK5000 | Next generation electroporation instrument |
| Neon Tranfection System 10 μl Kit | ThermoFisher Scientific | MPK1096 | |
| Resuspension Buffer T | ThermoFisher Scientific | Provided in kit (MPK1096) | "Transfection Resuspension Buffer" |
| Lymphoprep | Stemcell Technologies | #07801 | Density Gradient Medium |
| costar 6-well tissue culture treated plates | Corning | 3516 | flat bottom plates |
| costar 48-well tissue culture treated plates | Corning | 3548 | flat bottom plates |
| BD Pharm Lyse lysing buffer, 10 X | BD biosciences | 555899 | Must make 1X solution with distilled water prior to use |
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