荧光融合蛋白在小鼠骨髓基质树突状细胞和巨噬细胞中的表达
Summary
本文详细介绍了荧光融合蛋白在小鼠骨髓树突状细胞和巨噬细胞中的表达。该方法以骨髓祖细胞转导为基础, 采用逆转录病毒构造, 然后在体外分化成巨噬和树突状体细胞。
Abstract
树突状细胞和巨噬细胞是形成防御病原体的第一道防线的关键单元。它们也在启动自适应免疫反应中发挥重要作用。与这些细胞的实验工作是相当困难的。它们在器官和组织中的丰度相对较低。因此, 它们不能被大量隔离。它们也很难与 cDNA 构造转染。在小鼠模型中, 这些问题可以部分克服由骨髓祖细胞存在的细胞或 GM 脑脊液的树突状干细胞的体外分化。这样, 就可以从很少的动物那里获得大量这些细胞。此外, 骨髓祖细胞可以转导与逆转录病毒载体, 在其分化为骨髓衍生树突状干细胞和巨噬细胞之前, 在早期培养阶段进行 cDNA 构造。因此, 逆转录病毒转导后的体外分化可用于表达这些细胞中的各种 cDNA 结构。表达异位蛋白的能力大大扩展了可在这些细胞上进行的实验范围, 包括荧光蛋白的活细胞成像、路特分析的串联纯化、结构-功能分析、利用生物传感器和其他许多功能监测蜂窝细胞的机能。在本文中, 我们描述了一个详细的协议的逆转录病毒转导的小鼠骨髓衍生树突状细胞和巨噬细胞与载体编码的荧光标记蛋白。以两个适配器蛋白、OPAL1 和 PSTPIP2 为例, 说明其在流式细胞仪和显微术中的实际应用。我们还讨论了这种方法的优点和局限性。
Introduction
髓细胞是我们抵御病原体的防御机制不可或缺的一部分。他们能够迅速消除微生物, 以及垂死的细胞。此外, 他们还参与调节组织的发展和修复, 并保持稳态1,2,3。所有髓细胞均与骨髓中的常见髓内祖先分化。它们在许多功能和形态上不同的子集的分化是在很大程度上由细胞因子和它们的各种组合控制4。最深入研究的髓细胞亚群包括中性粒细胞粒细胞、巨细胞和树突状体细胞。任何这些人群中的缺陷会导致潜在的危及生命的后果, 并导致人类和小鼠免疫系统的严重功能失调1,2,3,5,6。
与中性粒细胞粒细胞不同的是, 树突状体细胞和巨噬细胞是组织的驻留单元, 免疫器官的丰度相对较低。因此, 在需要大量这些细胞的实验中, 分离和纯化原代树突状细胞和巨细胞是昂贵的, 而且往往是不可能的。为了解决这一问题, 已经开发出了一种在体外获得大量同质巨噬细胞或树突胞的实验方案。这些方法的基础是在细胞因子存在下的小鼠骨髓细胞分化: 巨噬细胞集落刺激因子 (M CSF) 用于巨噬细胞和粒细胞-巨噬 Flt3 集落刺激因子 (GM-csf) 或树突状细胞7,8,9,10,11,12。这种方法产生的细胞通常在文献中描述为骨髓衍生巨噬细胞 (BMDMs) 和骨髓衍生树突状细胞 (BMDCs)。它们与原生巨噬细胞或树突状干细胞的共同生理特性高于相应的细胞系。另一个主要优势是获得这些细胞形成转基因小鼠13的可能性。从转基因小鼠中提取的野生型细胞和细胞之间的比较研究对于揭示基因或感兴趣的蛋白质的新功能通常是至关重要的。
在活细胞中蛋白质的亚细胞定位分析需要荧光标签与体内感兴趣的蛋白质的耦合。这通常是通过表达由分析的蛋白质耦合 (通常通过短链接器) 与荧光蛋白 (例如, 绿色荧光蛋白 (GFP))14、15的基因编码的融合结构来实现的。,16. 荧光标记蛋白在树突状细胞或巨噬细胞中的表达具有挑战性。这些细胞通常难以转染的标准转染程序和效率往往是非常低的。此外, 转染是瞬态的, 它产生细胞应力和达到的荧光强度可能不足以显微镜17。为了获得足够的转基因表达水平的这些细胞的合理分数, 骨髓祖细胞与逆转录病毒载体的感染及其随后的分化成 BMDMs 或 BMDCs 已成为一个非常有效的方法。它允许对其原生细胞环境中的髓样蛋白进行分析, 无论是在稳态还是在对免疫反应至关重要的过程中, 如吞噬、免疫突触形成或迁移。在这里, 我们描述一个协议, 允许在小鼠骨髓巨噬细胞和树突状细胞中稳定表达荧光标记蛋白的兴趣。
Protocol
这里描述的所有方法都是由分子遗传学研究所的实验动物福利问题专家委员会和捷克共和国科学院批准的。 1. 试剂制备 制备氯化铵-钾 (ACK) 缓冲液。添加 4.145 g 的 NH4Cl 和 0.5 g 的 KHCO3到500毫升的 ddH2O, 然后添加100µL 的 0.5 M 乙酸酸 (EDTA) 和过滤消毒。 准备聚乙烯亚胺 (PEI) 解决方案。加入0.1 克 PEI 到90毫升的 ddH2O。搅拌时, 在 pH 值?…
Representative Results
信号适配器蛋白通常是小蛋白质, 没有任何酶活性。它们具有各种相互作用的领域或图案, 它调解与信号传导相关的其他蛋白质的结合, 包括酪氨酸激酶、磷酸酶、泛素连接酶等21。为演示此协议的功能骨髓细胞适配器 PSTPIP2 和 OPAL1 被选择。PSTPIP2 是一个很好的特征蛋白参与炎症反应的调节22。它是一种细胞质蛋白, 也可以通过其 F bar 域被…
Discussion
目标细胞中蛋白质的表达是许多生物研究的关键步骤。通过标准转染和逆转录病毒转导技术很难转染分化的巨噬细胞和树突状单元。绕过这些分化细胞的转染骨髓祖的逆转录病毒转导, 其次是分化时, 他们已经携带所需的结构, 是一个关键步骤, 允许在这些 cDNAs 的表达单元格类型。成功使用此方法的一个例子可以在我们最近的出版物25中找到。在这里, 我们提供了一个经济高效的协…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了捷克科学基金会 (GACR 项目编号 16-07425S) 的支持, 由查尔斯大学赠款机构 (GAUK) (项目编号 923116) 和捷克科学院分子遗传学研究所的机构资助。共和国 (RVO 68378050)。
Materials
| DMEM | Thermo Fisher Scientific, Waltham, MA, USA | 15028 | |
| Fetal bovine serum (FBS) | Thermo Fisher Scientific, Waltham, MA, USA | 10270 | For media suplementation |
| KHCO3 | Lachema, Brno, Czech Republic | N/A | |
| NH4Cl | Sigma-Aldrich (Merck, Kenilworth, NJ, USA) | A9434 | |
| Penicillin | BB Pharma AS, Prague, Czech Republic | N/A | PENICILIN G 1,0 DRASELNÁ SOL' BIOTIKA |
| Streptomycin | Sigma-Aldrich (Merck, Kenilworth, NJ, USA) | S9137 | Streptomycin sulfate salt powder |
| Gentamicin | Dr. Kulich Pharma, Hradec Králové, Czech Republic | N/A | |
| Polyethylenimine, linear, MW 25,000 | Polyscience, Warrington, PA, USA | 23966 | |
| Polybrene | Sigma-Aldrich (Merck, Kenilworth, NJ, USA) | H9268 | |
| EDTA | Sigma-Aldrich (Merck, Kenilworth, NJ, USA) | E5134 | |
| PBS | Prepared in-house by media facility of IMG ASCR, Prague, Czech Republic | N/A | |
| APC anti-mouse/human CD11b Antibody, clone M1/70 | BioLegend (San Diego, CA, USA) | 101212 | flow cytometry analysis |
| PE anti-mouse F4/80 Antibody, clone BM8 | BioLegend (San Diego, CA, USA) | 123110 | flow cytometry analysis |
| APC anti-mouse CD11c Antibody, clone N418 | BioLegend (San Diego, CA, USA) | 117310 | flow cytometry analysis |
| M-CSF | PeproTech (Rocky Hill, NJ, USA) | 315-02 | |
| GM-CSF | PeproTech (Rocky Hill, NJ, USA) | 315-03 | |
| Hoechst 33258 | Thermo Fisher Scientific, Waltham, MA, USA | H1398 | flow cytometry analysis use at 1-2 µg/ml |
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