RNA纯化在细胞内生长<em>李斯特菌</em>巨噬细胞
Summary
Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.
Abstract
Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.
Introduction
细胞内细菌病原体─引起传染病和能生长和再现人类宿主细胞内的细菌─是全世界主要的健康关注5。入侵和哺乳动物细胞内复制，细胞内病原体已取得成熟的致病机制和因素。虽然这些机制是引起疾病的能力根本，我们知之甚少的监管和动态。因为在液体培养基中生长的细菌的基因表达分布不反映宿主细胞内的实际的环境中，人们越来越需要转录在它们的细胞内的壁龛生长的细菌的分析。这种分析将使主机触发特异性细菌适应的解密，并将有助于确定治疗方案设计新的目标。细胞内生长的细菌的转录组分析是非常具有挑战性，因为哺乳动物RNA多于由细菌RNA至少十倍。在这个手稿中，我们描述的实验方法，从单核细胞增生李斯特菌小鼠巨噬细胞中分离出不断增长的细菌RNA。提取的RNA可用于研究细胞内的适应和致病菌转录分析的各种技术，如RT-PCR，RNA测序，微阵列和其它基于杂交的技术的毒力机制。
李斯特菌是李斯特菌在人体中的病原体，临床表现主要对象是免疫功能低下者，老人和孕妇6的疾病。它 是，侵入一个宽已在宿主-病原体的相互作用被使用了几十年作为模型研究7哺乳动物细胞的阵列的革兰氏阳性兼性细胞内病原体。在侵袭，它最初驻留在液泡或吞噬体（在吞噬细胞的情况下），从它必须转义入T他宿主细胞胞质溶胶中，以便进行复制。几个毒力因子已经显示介导的逃生过程中，主要是孔隙形成溶血素，李斯特O（LLO）和两个附加的磷脂8。在胞质溶胶中的细菌使用主机肌动蛋白聚合机械推动自己对小区内的肌动蛋白丝，并从细胞扩散到细胞（ 图1）。 L.所有主要毒力因子参与入侵，细胞内生存和复制的单核细胞 ，都是由主控毒力调节转录激活，经皮射频消融8-10。
在过去十年中，许多研究由我们进行的和其他人已经成功地应用于宿主细胞2,11-15内部为胞内生长的细菌的转录组分析的方法。两种主要的方法用于细菌RNA从其基于主机的RNA分离：1）细菌RNA的选择性富集和2）RNA分离由迪菲rential细胞裂解。第一种方法依赖于（使用市售的试剂盒为例）的总RNA提取物消减杂交哺乳动物RNA分子或细菌转录序列（SCOTS）11选择性捕获。第二种方法依赖于细菌和宿主细胞中，其中在细菌细胞中保持完整的宿主细胞裂解的差分裂解。细菌细胞，然后从宿主细胞裂解物中分离，通常是通过离心，并且将RNA是使用标准技术提取。使用这种方法的主要问题是，连同完好无损的细菌，宿主细胞细胞核也分离，从而将RNA制剂仍含有哺乳动物RNA。克服这个问题的一种方式是完整的细菌使用差速离心的宿主细胞的细胞核中分离出来，虽然这过程通常需要时间提高在基因表达谱变化的提取过程中的关注。在本文中，我们提出了一个改进的和快速的巴cteria RNA提取协议，这是基于对细胞差动裂解的方法。首先，L.巨噬细胞感染单核细胞的细胞裂解用冷水。接着，巨噬细胞的核是由一个简短离心除去完整细菌迅速收集在过滤器上，从该RNA被使用的细菌核酸的热酚-SDS提取隔离。
Protocol
Representative Results
Discussion
这里描述的协议代表了从属细菌RNA分离的优化方法细菌单核细胞巨噬细胞在细胞内不断增长。这个协议是基于细胞差动裂解，并包括用于细菌RNA富集两个主要步骤：使用离心和过滤细菌的快速采集巨噬细胞的细胞核沉淀。这些步骤之后是一个标准的RNA提取步骤。虽然这个协议描述李斯特菌RNA的纯化，它可以很容易地修改为其它的细菌病原体。虽然方法主要针对RNA进行转录分析的纯化?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
The research in the Herskovits lab is supported by 335400 ERC and R01A/109048 NIH grants.
Materials
| Listeria monocytogenes 10403S | 20 | ||
| Bone marrow derived macrophages prepared from C57B/6 female mice | 21 | ||
| H2O, RNAse free | Thermo Scientific | 10977-015 | DEPC-treated water can be used |
| DMEM | Gibco | 41965039 | |
| Glutamine | Gibco | 25030081 | |
| Sodium pyruvate | Gibco | 11360-088 | |
| b-Mercaptoethanol | Gibco | 31350010 | |
| Pen/Strep | Gibco | 15140-122 | |
| Gentamicin | Sigma-Aldrich | G1397 | |
| FBS | Gibco | 10270106 | |
| Dulbecco’s Phosphate Buffered Saline-PBS | Sigma-Aldrich | D8537 | |
| Brain heart infusion (BHI) | Merckmillipore | 1104930500 | |
| Phenol saturated pH 4.3 | Fisher | BP1751I-400 | |
| Chloroform | Fisher | BP1145-1 | |
| Iso-amyl alcohol | Sigma-Aldrich | W205702 | |
| Sodium acetate | Sigma-Aldrich | W302406 | |
| EDTA | Sigma-Aldrich | EDS | |
| DNaseI | Fermentas, | EN0521 | |
| SDS 10% | Sigma-Aldrich | L4522 | |
| Ethanol absolute | Merck Millipore | 1070174000 | |
| 37°C, 5% CO2 forced-air incubator | Thermo Scientific | Model 3111 | |
| Cell scrapers | Nunc | 179693 | |
| Kontes glass holder for 45 mm filters | Fisher | K953755-0045 | |
| MF-Millipore filters 45 mm, 0.45 µm | Merck Millipore | HAWP04700 | |
| SpeedVac system | Thermo Scientific | SPD131DDA | |
| Vortex-Genie 2 | Scientific Industries | Model G560E | |
| NanoDrop | Thermo Scientific | ||
| 145 mm cell culture dishes | Greiner | 639 160 | |
| 1.7 ml tubes, RNase-free | Axygen | MCT-175-C | |
| 30°C incubator | Thermo Scientific | ||
| 65 °C heat block | Thermo Scientific | ||
| 4 °C table centrifuge | Eppendorf | 5417R | |
| Sterile pipettes, 25 ml | Greiner | ||
| Falcon tubes, 50 ml | Greiner | ||
| Liquid nitrogen |
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