优化慢病毒生产和细胞培养必要条件，成功转导初级人支气管上皮细胞
Summary
Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.
Abstract
在使用空气-液体界面条件下提供了一个有用的模型来研究气道细胞分化和功能的处理原代人支气管上皮（HBE）细胞的体外培养。在过去的几年中，为转基因递送中使用的慢病毒载体的成为普遍的做法。虽然有完全分化的呼吸道上皮细胞与某些非HIV伪类型的慢病毒转导的报道，整体转导效率通常低于15％左右。这里介绍的协议提供了可靠和有效的方法，以产生慢病毒和转导原代人支气管上皮细胞。用未分化的支气管上皮细胞，支气管上皮生长培养基转导，而细胞附着，具有4感染因子的多个提供接近100％的效率。本协议描述的，一步一步的，高滴度的慢病毒载体及日的准备和浓度Ë传导过程。它讨论了确定的最佳培养条件，以实现初级人支气管上皮细胞的高效转导的实验。
Introduction
复制缺陷型，假慢病毒的发展（LV）也为研究人员提供一个安全和有效的方法， 在体外 1提供转基因。由于它们的长期表达，这些LV也可以用于治疗剂的体内 2,3的输送。生产的LV需要人胚胎肾（HEK）细胞的几个质粒共转染：转移载体，包装的gag / POL，包装转速 ，和包络水泡性口炎病毒糖蛋白（VSV-G）的质粒。第三代包装系统被认为比第二代系统更安全，因为转速基因被编码在一个单独的包装质粒，从而降低重组的可能性，以产生具有复制能力的慢病毒。虽然第二代包装系统收率五倍高的总转导单元，原病毒基因组的分割以创建第三代系统已降低转导的水平仅最低限度3,4。
而细胞系可更容易和有效地转导，研究人员已经将重点转向主细胞，因为这些是更具有代表性的体内组织中5,6。然而，原代细胞是难治转导。而完全分化呼吸道上皮细胞的转导已经报道，整体效率还没有超过15％的7。
这里描述的是一个协议，允许生产高滴度的LV的。一个一步一步的方法概述实现近100％的转导效率分为原发性支气管上皮细胞。更具体地，该协议描述器乐成功3的最佳培养条件。简言之，HEK 293T细胞使用的是带有增强型绿色荧光的EF-1启动子驱动表达慢病毒载体转染PCDH蛋白（EGFP）或mCherry，红色荧光蛋白，和一个第三代包装系统。释放到介质的LV被收集24至72小时后。病毒颗粒集中用聚乙二醇（PEG），病毒浓度的方便和简单的方法，采用了p24抗原的酶联免疫吸附测定（ELISA）试剂盒滴度估计之前。随后，未分化的初级HBE细胞被转导在增殖培养基（支气管上皮生长培养基或BEGM）8，而安装（被胰蛋白酶处理后），在感染复数的4（MOI）因子，加入聚凝胺和温育16小时（过夜）。然后允许细胞分化。由于病毒的转基因被表达长期（使用适当的启动子，见讨论），表达将在整个分化维持成假呼吸道上皮或可以（使用诱导型启动子）的分化后诱导。
<p cl屁股="“jove_content”">此协议是广泛的兴趣谁想要使用原HBE细胞，而不是细胞系的研究人员，并可能适用于其他难以转导的细胞类型。Protocol
Representative Results
Discussion
这里所概述的协议保证接近100％转导效率。但是，也有在此过程中是实现这一目标的重要步骤。例如，在转染过程中，析出物中的转导结构的存在（下降），或在培养皿后转导（ 图1b和d）该天是一个良好的转染都相关。没有沉淀物或附聚物的存在可能是由于在转染试剂中的一个的遗漏，pH值的问题，或坏质粒制备。如果沉淀缺失，有很高的可能性，没有将可见的第二天。…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们感谢迈阿密大学的生命器官联盟追回署提供的肺部。我们也感谢丽莎昆兹博士用于图2B和3-D，本Gerovac博士和丽莎·诺瓦克在图3A至C.用于实验的mCherry病毒所示的实验DNA准备，我们也感谢加布里埃尔Gaidosh从成像设备，眼科在迈阿密大学。
这项研究是由来自美国国立卫生研究院，在CF基金会和乘务员医学研究所的马蒂亚斯Salathe博士助学金资助。
Materials
| HEK293T/17 cells | ATCC | CRL-11268 | |
| Fetal bovine serum (FBS) | Sigma | 12306C | use at 10% |
| DMEM | Thermo Scientific | 11995-040 | |
| Penicillin/Streptomycin (100x) | Thermo Scientific | 15140-122 | use at 1x |
| Collagen I | BD Biosciences | 354231 | dilute 1:75 in distilled water |
| Calphos | Clontech | 631312 | Transfection kit containing 2M Calcium solution, 2X HEPES-Buffered solution (HBS) and sterile H2O |
| Polythylene glycol 8000 | VWR scientific | 101108-210 | stock of 40% |
| Amphotericin B | Sigma | A9528 | use at 1x |
| Trypsin | Sigma | T4799 | |
| Soybean trypsin inhibitor | Sigma | T9128 | |
| Transwell 12mm | Corning | 3460 | |
| Collagen IV | Sigma | C7521 | dilute 1:10 in distilled water |
| Hexadimethrine bromide | Sigma | H9268 | stock of 2mg/ml |
| Puromycin (10.000x) | Thermo Scientific | A11138-03 | use at 1x |
| Alliance HIV-1 p24 ELISA | PerkinElmer | NEK050001KT | |
| F12 | Thermo Scientific | 11765-054 | |
| pCDH-EF1-MCS-IRES-Puro | System Biosciences | CD532A-2 | lentiviral expression vector |
| pMD2-VSVG | Addgene | 12259 | packaging DNA |
| pMDLg/pRRE | Addgene | 12251 | packaging DNA |
| pRSV-rev | Addgene | 12253 | packaging DNA |
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