Name: using rna-interference to investigate the innate immune response in mouse macrophages
Uploaded: 2014-11-03T13:00:00
Description: Watch this Scientific Journal Video about Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages at JoVE.com

Thanks to Brad Lackford for assistance optimizing some of the techniques described in this manuscript.

マクロファージ細胞株の1メンテナンス<ol><li> RAW264.7を成長させ、DMEM中J774A.1細胞株は、5％CO 2存在下、37ºCで10％ウシ胎児血清（FBS）を補充した。 （これは厳密には必要ではないが）無菌性を維持するために、メディアへの1％ペニシリン/ストレプトマイシン（ペニシリン/ストレプトマイシン）を追加します。 </li><li> 重要なステップ：マクロファージは効率的なトラン?...

<ol>
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A., Nilsson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lysozyme+synthesis+by+established+human+and+murine+histiocytic+lymphoma+cell+lines.">Lysozyme synthesis by established human and murine histiocytic lymphoma cell lines.</a> The Journal of experimental medicine. , 1528-1533 (1976).</li><li>Zumbansen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+siRNA+library+screening+in+hard-to-transfect+HUVEC+cells.">First siRNA library screening in hard-to-transfect HUVEC cells.</a> Journal of RNAi and gene silencing. 6, 354-360 (2010).</li><li>Lu, Y. C., Yeh, W. C., Ohashi, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LPS/TLR4+signal+transduction+pathway.">LPS/TLR4 signal transduction pathway.</a> Cytokine. 42, 145-151 (2008).</li><li>Aliprantis, A. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+activation+and+apoptosis+by+bacterial+lipoproteins+through+toll-like+receptor-2.">Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2.</a> Science. 285, 736-739 (1999).</li><li>Alexopoulou, L., Holt, A. C., Medzhitov, R., Flavell, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+double-stranded+RNA+and+activation+of+NF-kappaB+by+Toll-like+receptor+3.">Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3.</a> Nature. 413, 732 (2001).</li><li>Conforti, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+effects+of+toll-like+receptor+(TLR3)+signaling+in+tumors+can+be+therapeutically+uncoupled+to+optimize+the+anticancer+efficacy+of+TLR3+ligands.">Opposing effects of toll-like receptor (TLR3) signaling in tumors can be therapeutically uncoupled to optimize the anticancer efficacy of TLR3 ligands.</a> Cancer Res. 70, 490-500 (2010).</li><li>Fernandez-Botran, R., V&#283;tvi&#269;ka, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Cellular Immunology. 8, 8 (2001).</li></ol>

多くの研究は、個々の遺伝子は、マウスのマクロファージにおけるsiRNAの標的とされている中で発表されている。脂質媒介トランスフェクションが個別にマクロファージ細胞株に対するsiRNAを送達するために使用されてきたが、これらの方法は、生存能力、限られた遺伝子ノックダウン、および遺伝子からの遺伝子の可変性に対する潜在的な影響に苦しんでいる。中·高スループット様式で遺?...

このプロトコルでは、siRNAを用いてマウスマクロファージ細胞株における遺伝子を抑制し、自然免疫応答におけるこれらの治療の効果をモニターするために効率的な方法を記載する。これらの手順は、中または高スループット様式でRNAiスクリーニングを可能にする、96ウェル形式で行われる。 感染に応答して、人間が直接の先天性免疫応答、より遅いが、より具体的な適応免疫応答をマウントする。この急速な自然免疫応答は、マクロファージ1を含む貪食自然免疫細胞の動員および活性化を伴う。古典的に活性化されたマクロファージは、急性炎症反応に関与し、抗菌タンパク質および化合物、サイトカイン及びケモカイン、および抗原を提示2,3を生成している。あるいは、活性化マクロファージは公差、組織修復を維持し、免疫を調節する役割を果たし、4-8創傷治癒 。理由の機能の彼らの広い配列のマクロファージは、アテローム性動脈硬化症、関節炎、および癌9を含む多数の疾患において役割を果たすことができる。したがって、マクロファージの研究では、病気の幅広い分野でいくつかの時間のための研究の重要な分野となっている。 先天性免疫応答におけるその重要性にもかかわらず、マクロファージはで動作するように細胞に挑戦することができます。特に、関連する毒性10,11なしマクロファージにおける脂質試薬を用いて効率的なトランスフェクションを得ることは困難である。 siRNAは効果的にマクロファージに送達される場合にもまた、RNAiを誘導遺伝子ノックダウンの頑健性は、多くの場合、かなり中程度であることができ、遺伝子に遺伝子を変化させることができる。 これらの技術的課題を克服するために、我々は、2つのマウスマクロファージ細胞株RAW264.7 17とJ774A.1 18でトランスフェクションし、ノックダウン技術12-16を最適化した。このアプローチは、トランスフェクションのためにAmaxa社のNucleofector 96ウェルシャトルシステムを使用しています。このシステム96ウェルフォーマット19で細胞をトランスフェクトするための特殊な試薬やエレクトロポレーションを組み合わせて使用します。トランスフェクションの後、我々は、細胞の回収および生存率を最大化するために、後続のsiRNAによる遺伝子ノックダウンを最大化するための方法を記載している。このアプローチの有用性を説明するために、リポ多糖（LPS）でこれらの細胞を刺激し、いくつかの炎症誘発性サイトカインの産生のレベルにおける先天性免疫応答をモニターし、これらのマクロファージ細胞株へのsiRNA送達のためのプロトコルを記述している。私たちはメンバー先天性免疫を調節するために、LPS及び他の病原体関連分子パターン（PAMP）を感知し、Toll様受容体（TLR）ファミリーをターゲットとしたサンプルデータを提供する。

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >Amaxa nucleofector 96-well shuttle system</td>
			<td>Lonza</td>
			<td>AAM-1001S</td>
		<td></td></tr>
		<tr >
			<td >Amaxa SF cell line 96-well nucleofector kit</td>
			<td>Lonza</td>
			<td>V4SC-2096</td>
		<td></td></tr>
		<tr >
			<td >RAW264.7 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-71</td>
		<td></td></tr>
		<tr >
			<td >J774A.1 mouse macrophage cell line</td>
			<td>ATCC</td>
			<td>TIB-67</td>
		<td></td></tr>
		<tr >
			<td >siGenome smartpool siRNA</td>
			<td>Dharmacon</td>
			<td>varies depending on gene</td>
		<td></td></tr>
		<tr >
			<td >Non-targeting control siRNA pool</td>
			<td>Dharmacon</td>
			<td>D-001206-13-20</td>
		<td></td></tr>
		<tr >
			<td >Block-iT fluorescent oligo for electrooration</td>
			<td>Invitrogen</td>
			<td>13750062</td>
		<td></td></tr>
		<tr >
			<td >Ultrapure E. coli O111:B4 LPS</td>
			<td>List Biological Laboratories</td>
			<td>421</td>
		<td></td></tr>
		<tr >
			<td >DMEM, high glucose</td>
			<td>Invitrogen</td>
			<td>11995-065</td>
		<td></td></tr>
		<tr >
			<td >RPM1-1640</td>
			<td>Invitrogen</td>
			<td>11875-093</td>
		<td></td></tr>
		<tr >
			<td >Penicillin-Streptomycin Solution (Pen/Strep)</td>
			<td>Fisher</td>
			<td>SV30010</td>
		<td></td></tr>
		<tr >
			<td >0.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200072</td>
		<td></td></tr>
		<tr >
			<td >96 well tissue culture plates</td>
			<td>Fisher</td>
			<td>07-200-89 </td>
		<td></td></tr>
		<tr >
			<td >96 well round bottom sterile plates (not coated)</td>
			<td>Fisher</td>
			<td>07-200-745</td>
		<td></td></tr>
		<tr >
			<td >Mouse IL-6 Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY406</td>
		<td></td></tr>
		<tr >
			<td >Mouse TNFa Duoset ELISA kit</td>
			<td>R&#38;D Biosystems</td>
			<td>DY410</td>
		<td></td></tr>
		<tr >
			<td >Fluorescein diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>F7378</td>
		<td></td></tr>
		<tr >
			<td >RLT Bufer</td>
			<td>Qiagen</td>
			<td>79216</td>
		<td></td></tr>
		<tr >
			<td >Rneasy mini kit</td>
			<td>Qiagen</td>
			<td>74134</td>
		<td></td></tr>
		<tr >
			<td >Vybrant Phagocytosis Assay Kit</td>
			<td>Invitrogen</td>
			<td>V-6694</td>
		<td></td></tr>
	</tbody>
</table>

using rna-interference to investigate the innate immune response in mouse macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

このアプローチを使用するトランスフェクションの効率を実証するために、我々は、フローサイトメーター（ 図1）を用いてFITC標識siRNAの取り込みを監視した。 先天性免疫応答をモニターするための我々のアプローチの有用性を説明するために、我々は、RAW264.7マクロファージ細胞株に知ら自然免疫調節遺伝子を標的とするsiRNAをトランスフェクトされたLPSで...

Watch this Scientific Journal Video about Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages at JoVE.com

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

マウスマクロファージにおける先天性免疫応答を調査するために、RNA干渉を使用

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Research

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ダニのRNA干渉

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

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An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

の寄生蜂の紹介<em&gt;ショウジョウバエ</em&gt;と抗寄生虫免疫応答

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

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Quantitative Measurement of the Immune Response and Sleep in Drosophila

免疫応答やスリープ中の定量的測定<em&gt;ショウジョウバエ</em

A complex interaction between the immune response and  host behavior has been described in a wide range of species. Excess sleep, in  particular, is known ...

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ゼブラフィッシュにおける自然免疫の健康の指標として呼吸バースト応答の定量化

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

サイトカイン産生のウイルス回避における先天性免疫シグナルを解剖

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

In Vitro Analysis of Myd88-mediated Cellular Immune Response to West Nile Virus Mutant Strain Infection

西ナイルウイルス変異株の感染に対するMyD88非介在細胞性免疫応答のインビトロ分析

An attenuated West Nile virus (WNV), a nonstructural (NS) 4B-P38G mutant, induced higher innate cytokine and T cell responses than the wild-type WNV in ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

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のゼブラフィッシュ幼生モデルにおける先天性免疫応答の非侵襲的イメージング<em&gt;ストレプトコッカスイニエ</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

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系統解析を使用して真核生物遺伝子の起源を調査するには

Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

リポ多糖投与マウス肝臓における免疫応答時に白血球の二光子励起生体イメージング

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...