Name: highly efficient transfection of primary macrophages with in vitro transcribed mrna
Uploaded: 2019-11-09T13:00:00
Description: Watch this Scientific Journal Video about Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA at JoVE.com

这项工作得到了德国福森斯金舍夫（SFB 670）的支持。

根据德国《动物保护法》，根据科隆大学伦理委员会对小鼠进行巨噬菌体分离。
注：戴上手套执行所有步骤。在层流罩下执行所有转染步骤，以防止细胞受到污染。在使用 mRNA 之前，使用 70% 乙醇和/或 RNAe 降解表面活性剂（材料表）清洁所有仪器，如移液器和每个表面。确保所有反应管无RNA酶和无菌。只使用无菌的无RNA酶水进行稀释。专门使用带过?...

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在这里，我们提出了一个协议，用于使用体外转录mRNA对通常难以转染的通常难以转染的初级巨噬细胞进行高效转染。重要的是，使用该协议的巨噬细胞转染不会诱发细胞死亡或激活亲炎信号，表明转染试剂和转染的mRNA均不被识别为非自体。
mRNA的质量对于使用该协议成功转染巨噬细胞至关重要。因此，应非常小心，mRNA不会与RNA接触（例如，通过受污染的缓冲液或小瓶）。如?...

巨噬细胞是噬菌体，专门检测、摄取和降解微生物、凋亡细胞和细胞碎片。此外，它们通过分泌细胞因子和化学因子，以及向T细胞和B细胞提供抗原，帮助协调免疫反应。巨噬细胞在许多其他过程中也起着重要作用，如伤口愈合、动脉粥样硬化、肿瘤发生和肥胖。
为了能够检测非自分子，如病原体相关的分子模式（PAMPs）和不合时宜的分子，如损伤相关分子模式（DAMPs），巨噬细胞配备了大量模式识别受体（PRR）1。不幸的是，这也使得巨噬细胞特别难以转染2作为转染试剂3和转染核酸4，5，6，7经常被PR识别为非自体。因此，使用化学或物理方法的巨噬细胞转染通常会导致大噬细胞活化和转染核酸的降解，甚至导致巨噬细胞通过热膜病自杀，这是一种在识别细胞环管PAMPs/DAMPs（如DNA或外来RNA9）后触发的细胞细胞死亡。使用腺病毒或扁病毒等病毒作为载体的巨噬细胞的生物转染通常效率更高，但构建这种病毒载体却非常耗时，需要生物安全2级设备10、11。
因此，虽然巨噬细胞是深入研究的主题，但由于分子生物学最重要的工具之一，核酸构造的转染，用于外源性表达，因此在分子水平上对其功能的分析受到了阻碍。蛋白质，是很难适用的。这经常迫使研究人员使用类似巨噬细胞的细胞系，而不是真正的巨噬细胞。核酸构造转染的应用包括突变或标记蛋白的表达、特定蛋白质的过度表达、在各自敲除背景中的蛋白质重新表达以及来自其他物种的蛋白质的表达（例如。，Cre 重组酶或指导 RNA 和 Cas9 用于靶向基因敲除）。
在这里，我们描述了一个协议，允许高效转染（通常难以转染）原体噬菌体，即鼠围管巨噬细胞（PM）和骨髓衍生巨噬细胞（BMDM）与MRNA产生的DNA模板，如质粒。重要的是，使用该协议产生的体外转录mRNA含有自然产生的修饰核苷5-甲基-CTP和伪UTP，降低免疫原性，增强稳定性4，6，7，12，13。此外，在体外转录的mRNA的5'端被南极磷酸酶去磷化，以防止RIG-I复合体14、15的识别。这最大限度地减少了体外转录mRNA的先天免疫识别。通过我们易于执行的协议，转染率在50-65%（围脑巨噬细胞（PM））和85%（BMDM）之间，而重要的是，没有观察到细胞毒性或免疫原性。我们详细描述 （i） 如何从DNA结构（如质粒）和 （ii） 转染程序本身生成用于转染的免疫沉默的 mRNA。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5-methyl-CTP (100 mM)</td>
			<td>Jena Biosience</td>
			<td>NU-1138S</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Antarctic phosphatase</td>
			<td>New England BioLabs</td>
			<td>M0289</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Antarctic phosphatase reaction buffer (10X)</td>
			<td>New England BioLabs</td>
			<td>B0289</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>anti-NEMO/IKK&#947; antibody</td>
			<td>Invitrogen</td>
			<td>MA1-41046</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>anti-&#946;-actin antibody</td>
			<td>Sigma-Aldrich</td>
			<td>A2228</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Petri dishes 92,16 mm with cams</td>
			<td>Sarstedt</td>
			<td>821,473</td>
			<td>stored at RT</td>
		</tr>
		<tr>
			<td>CD11b Microbeads mouse and human</td>
			<td>Miltenyi Biotec</td>
			<td>130-049-601</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Cre recombinase + T7-Promotor forward primer</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>5&#8242;-GAAATTAATACGACTCACTATA GGGGCAGCCGCCACCATGTCC AATTTACTGACCGTAC-3&#180;, stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Cre recombinase + T7-Promotor reverse primer</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>5&#8242;-CTAATCGCCATCTTCCAGCAGG C-3&#8242;, stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>DNA purification kit: QIAquick PCR purification Kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>stored at RT</td>
		</tr>
		<tr>
			<td>eGFP + T7-Promotor forward primer</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>5&#180;-GAAATTAATACGACTCACTATA GGGATCCATCGCCACCATGGTG AGCAAGG-3&#180;, stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>eGFP + T7-Promotor reverse primer</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>5&#180;-TGGTATGGCTGATTA TGATCTAGAGTCG-3&#180;, stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Fast Digest buffer (10X)</td>
			<td>Thermo Scientific</td>
			<td>B64</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>FastDigest XbaI</td>
			<td>Thermo Scientific</td>
			<td>FD0684</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>high-fidelity polymerase with proofreading: Q5 High-Fidelity DNA-Polymerase</td>
			<td>New England Biolabs Inc</td>
			<td>M0491S</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>IKK&#946; + T7-Promotor forward primer</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>5&#8242;-GAAATTAATACGACTCACTATA GGGTTGATCTACCATGGACTACA AAGACG-3&#8242;, stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>IKK&#946; + T7-Promotor reverse primer</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>5&#8242;-GAGGAAGCGAGAGCT-CCATCTG-3&#8242;, stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>in vitro mRNA transcription kit: HiScribe T7 ARCA mRNA kit (with polyA tailing)</td>
			<td>New England BioLabs</td>
			<td>E2060</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-401</td>
			<td>stored at RT</td>
		</tr>
		<tr>
			<td>MACS MultiStand</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-303</td>
			<td>stored at RT</td>
		</tr>
		<tr>
			<td>mRNA transfection buffer and reagent: jetMESSENGER</td>
			<td>Polyplus transfection</td>
			<td>409-0001DE</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Mutant IKK&#946; IKK-2S177/181E plasmid</td>
			<td>Addgene</td>
			<td>11105</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Mutant NEMOC54/347A plasmid</td>
			<td>Addgene</td>
			<td>27268</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>pEGFP-N3 plasmid</td>
			<td>Addgene</td>
			<td>62043</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>poly(I:C)</td>
			<td>Calbiochem</td>
			<td>528906</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>pPGK-Cre plasmid</td>
			<td></td>
			<td></td>
			<td>F. T. Wunderlich, H. Wildner, K. Rajewsky, F. Edenhofer, New variants of inducible Cre
recombinase: A novel mutant of Cre-PR fusion protein exhibits enhanced sensitivity and
an expanded range of inducibility. Nucleic Acids Res. 29, 47e (2001). stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>pseudo-UTP (100 mM)</td>
			<td>Jena Biosience</td>
			<td>NU-1139S</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>QuadroMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-976</td>
			<td>stored at RT</td>
		</tr>
		<tr>
			<td>Rat-anti-mouse CD11b antibody, APC-conjugated</td>
			<td>BioLegend</td>
			<td>101212</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Rat-anti-mouse F4/80 antibody, PE-conjugated</td>
			<td>eBioscience</td>
			<td>12-4801-82</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>recombinant M-CSF</td>
			<td>Peprotech</td>
			<td>315-02</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>RNA purification kit: MEGAclear transcription clean-up kit</td>
			<td>ThermoFisher Scientific</td>
			<td>AM1908</td>
			<td>stored at 4 &#176;C</td>
		</tr>
		<tr>
			<td>RNAse-degrading surfactant: RnaseZAP</td>
			<td>Sigma-Aldrich</td>
			<td>R2020</td>
			<td>stored at RT</td>
		</tr>
		<tr>
			<td>ultrapure LPS from E.coli O111:B4</td>
			<td>Invivogen</td>
			<td></td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Wild type IKK&#946; plasmid</td>
			<td>Addgene</td>
			<td>11103</td>
			<td>stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Wild type NEMO plasmid</td>
			<td>Addgene</td>
			<td>27268</td>
			<td>stored at -20 &#176;C</td>
		</tr>
	</tbody>
</table>

highly efficient transfection of primary macrophages with in vitro transcribed mrna

巨噬细胞是专门检测非自源分子的噬菌体。为此，他们配备了大量模式识别受体（PRRs）。不幸的是，这也使得巨噬细胞特别难以转染，因为转染试剂和转染的核酸经常被PR识别为非自体。因此，转染通常会导致巨噬细胞活化和转染核酸的降解，甚至导致巨噬细胞的自杀。在这里，我们描述了一个协议，它允许高效转染鼠原性巨噬细胞，如围肠巨噬细胞（PM）和骨髓衍生巨噬细胞（BMDM），在体外转录从DNA模板（如质粒）的mRNA。通过这种简单的方案，在未观察到细胞毒性或免疫原性的情况下，PM和BMDM的转染率约为50-65%，BMDM约为85%。我们详细描述了从DNA结构（如质粒和转染过程）转染的mRNA的生成。

我们已经成功地使用该协议为FLAG标记的NEMO和IKK+变体生成mRNA编码，用于转染原巨噬细胞16。FLAG 标记野生型 （NEMOWT） 和 C54/347A 突变 NEMO （NEMOC54/374A）（见材料表）的质粒编码已经包含方向正确的 T7 前电机 （图 1A）。因此，我们只需要线性化质粒，以生成体外转录的DNA模板。为此，用5μL Xb...

Watch this Scientific Journal Video about Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA at JoVE.com

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

巨噬细胞，特别是原发性巨噬细胞，由于它们专门检测非自源分子，因此很难转染。我们描述了一种协议，它允许使用从DNA模板（如质粒）生成的mRNA对原性巨噬细胞进行高效的转染。

具有体外转录mRNA的初级巨噬细胞的高效转染

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...

由于巨噬细胞专门检测非自源性分子，它们特别难以转染。我们描述了一种协议，它允许利用从DNA模板（如质粒）生成的mRNA对原食巨噬细胞进行高效的转染。我们方法的主要优点是，在没有任何可检测的细胞毒性或免疫原性的情况下，可以达到高转染率。演示这个程序将是马克赫伯从我的实验室博士后。首先解冻RNA转录试剂盒的所有成分。将试剂旋出5秒钟，以2000倍g旋转两秒钟，然后将它们放在冰上直到使用。根据稿稿说明在0.5微升微离心管中准备体外RNA转录反应。旋转管，然后旋转下来。然后在37摄氏度下孵育30分钟，同时在热混合器上以400转/分混合。孵育后，将DNase I的两微升直接加入反应混合物中。涡流和旋转管下来，并在热混合器中孵育15分钟。为控制凝胶保留 Dnase 处理产品的两微升等分，并将其储存在零下 80 摄氏度。对于聚A尾矿，在Dnase处理的反应中加入5个10X聚A聚合酶反应缓冲液和5微升聚A聚合酶。漩涡和旋转下来的混合。然后在热混合器中孵育30分钟。当反应完成后，为控制凝胶服用两微升等分，并将其储存在零下80摄氏度。根据手稿说明，将 5 个主要脱磷酸酯试剂直接添加到聚A尾产品中。涡流和旋转下管，并在热混合器中孵育30分钟。遵循脱磷反应，在80摄氏度下孵育两分钟，以加热灭活南极磷酸酶。然后使用专用的RNA纯化试剂盒纯化体外转录mRNA。使用微卷分光光度计测量洗样mRNA的浓度和纯度。使用先前收集的等分，运行变性阿加罗斯凝胶，以验证单个产品是否存在、正确的转录长度和聚A尾随。通过将 mRNA 转染缓冲液的预计算体积减去 mRNA 转染试剂和 mRNA 的体积添加到反应管中，为转染做好准备。解冻 mRNA 库存，通过翻转管子轻轻混合。然后将mRNA的预计算体积添加到反应管中。涡流并旋转反应混合物和mRNA转染试剂，然后将mRNA转染试剂的适当体积添加到反应混合管中。旋转管，然后旋转下来。然后在室温下孵育15分钟。同时，用清新温暖的培养媒介代替巨噬细胞的培养媒介。转染混合物的孵育完成，将混合物加入含有巨噬细胞的板井中，并在井外到井中间的圆圈中加入。确保转染混合物的均匀分布，以垂直和水平方向轻轻摇动板。然后在37摄氏度和5%的二氧化碳下孵育至少6小时。孵育后，用荧光显微镜、流式细胞仪或免疫布洛特分析转染效率。这个程序最重要的部分是确保转染组合的均匀分布，使所有巨噬细胞接触相同数量的mRNA。该协议成功地用于生成 mRNA 编码 FLAG 标记 NEMO 和 IKK-beta 变种，用于原发巨噬细胞的转染。通过红糖凝胶电泳验证了mRNA的正确尺寸和聚A尾随。通过生成mRNA编码GFP并进行流细胞学分析，证实了转染效率。转染率随转染mRNA的量而增加，PM达到50%至65%，BMDM达到80至85%。在PM和BMDM中，转染细胞中的EGFP表达水平以时间和剂量依赖的方式增加。重要的是，对碘化阳性或附件V阳性巨噬细胞的分析证实，转染程序没有诱发细胞解或凋亡细胞死亡。利用免疫荧光显微镜对编码 FLAG 标记的尼莫或 IKK-beta 变种的 mRNA 的转染效率进行了分析。尼莫 mRNA 的费率约为 60%，IKK-beta mRNA 的费率约为 55%。该协议还用于生成 mRNA 编码 Cre 重组酶。40万BMDM转染与400纳米的Cre重组酶mRNA导致48小时后尼莫蛋白几乎完全耗尽，表明高效的转染。没有任何可检测量的白细胞素1β、白细胞素-6和肿瘤坏死因子表明转染程序不会激活亲炎信号。我们相对简单的方法最终允许有效地表达原发巨噬细胞中的突变或标记蛋白。这将大大进一步分析分子水平上的巨噬细胞功能。

Research

JoVE Journal

Immunology and Infection

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

一种简单而有效的隔离混合原代培养成人肝细胞巨噬细胞的方法

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

科研

免疫与感染

Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation

高效转染树突状细胞的无细胞的成熟优化的协议

Dendritic cells (DCs) can be considered sentinels of the immune system which play a critical role in its initiation and response to infection1. Detection ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

建立一个<em&gt;在体外</em&gt;系统来研究细胞内的行为<em&gt;光滑念珠菌</em&gt;在人THP-1巨噬细胞

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

量<em在体外</em&gt;激活的主人微血管内皮细胞在静态条件下测量的检测，以中性粒细胞粘附

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection

人THP-1巨噬细胞通过核转高效转染

Macrophages, as key players of the innate immune response, are at the focus of research dealing with tissue homeostasis or various pathologies. ...

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

Quantification of Cytosolic vs. Vacuolar Salmonella in Primary Macrophages by Differential Permeabilization

胞浆的定量与液泡<em&gt;沙门氏菌</em&gt;在原发性巨噬细胞差透

Intracellular bacterial pathogens can replicate in the cytosol or in specialized pathogen-containing vacuoles (PCVs). To reach the cytosol, bacteria like ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

吞噬体偏移和速度与HIV-1感染的活小学的人巨噬细胞测量

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

使用的淋巴细胞外渗分析<em&gt;体外</em&gt;人类血脑屏障模型

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

小RNA转染原代人Th17细胞通过下一代电穿孔

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...