この研究は、NIH R00HL102154によってサポートされ、P智への血液学者賞のアメリカの社会ました。

このプロトコルに記載される実験は、ノースウェスタン大学施設内動物管理使用委員会によって定められたガイドラインおよび規制に従って行った。 培養液の調製<ol><li>フィブロネクチン溶液を調製する。ヒトフィブロネクチン（1mg）を1バイアルに1mlの水を追加します。撹拌せずに30分間組織培養フード内の溶液のままにしておきます。 20μg/ mlの最終濃度を作るために、PBS 50mlに全液体を移し、静かによく混ぜる。プレートを被覆する前に、フィブロネクチン溶液は新鮮行う（下記参照）。 </li><li>エポ培地（SCF培地）50mlのを準備します。新鮮作ったとき、表1に列挙した成分を組み合わせる。培地を1ヶ月間4℃で保存することができる。 </li><li>エポ含む培地50mlを準備します。新鮮作ったとき、表2に列挙した成分を組み合わせる。培地を1ヶ月間4℃で保存することができる。 </li></オール&gt; 2フィブロネクチンコートプレート<ol><li> 6ウェルプレート（または12ウェルプレートの各ウェルに0.5 ml）を各ウェルに1 mlのフィブロネクチン溶液を添加する。 1時間の組織培養フード内でプレートにしておきます。 </li><li>二回滅菌水でウェルを洗浄し、空気プレートを乾燥させます。 </li><li>プレートをラップし、4℃で低温室で保存してください。 </li></ol>胎児肝臓赤芽球の精製注：E13からE15時限妊娠マウス（C57BL / 6）に使用して胎児の肝臓。 E13.5肝臓はCFU-E前駆体の収量を最大にするために好ましい。時限妊娠マウスを得るためには、6〜8週齢の雄と雌のマウスは1ケージ（1つまたは2つの雌と通常1オス）に入れた。女性の膣のプラグは交尾が成功したかどうかを判断するために次の日の朝にチェックした。妊娠の最初の日は、プラグが検出された後の一日でした。 <ol><li>総胎児肝細胞の調製<ol><li>畝を生け贄に捧げるCO 2吸入と頸椎脱臼によってSE。消毒用70％エタノールで腹部をスプレーします。子宮を除去するために、はさみを解剖して、腹部を開きます。 1×PBS中で子宮を洗ってください。 </li><li>組織培養フード、オートクレーブのツールを使用して無菌条件下で胎児を解剖し、1×PBS中で洗浄します。 </li><li>胎児からの胎児の肝臓を解剖。別の鉗子で身体から離れ胎児肝臓（色はピンク）をゆっくりと引い1ピンセットで胎児のボディを持ってください。関連する線維性組織を除去するための鉗子で胎児の肝臓を清掃してください。 10％ウシ胎児血清を含む新鮮な1×PBS中にすべての胎児の肝臓を置きます。 </li><li>機械的ピペッティングにより胎児の肝臓を混乱させる。 </li><li> 50mlの円錐チューブに40μmのセルストレーナーを通して細胞懸濁液をフィルタリングします。 5分間800×gで細胞をペレット化。 </li><li> FBSの上清を吸引し、10％1mlのPBSで細胞を再懸濁（約8 E13.5胎児肝臓）1mlあたり。 </li></ol></li><li> TER119陰性赤芽球の精製<ol><li>氷上で15分間、ラットのIgG（0.2 mg / ml）を用いて細胞をブロックする。 </li><li>洗浄又は遠心分離せずに、細胞懸濁液（1μg/ ml）をビオチン化抗TER119抗体を加える。氷上で15分間インキュベートする。 </li><li> 5分間800×gで、10％FBS（1:10）と遠心分離機に1×PBS中で洗浄する。 10％FBSを含む1mlのPBSで細胞を再懸濁する。 </li><li>磁性粒子とコンジュゲート75μlのストレプトアビジンを加え、氷上で10分間インキュベートする。 </li><li> PBSで10％FBSを含む2.5ミリリットルにボリュームを追加します。ボトムチューブラウンド5ミリリットルのポリプロピレンに細胞懸濁液を転送します。 </li><li>磁気細胞選別装置内にチューブを挿入し、10分間インキュベートする。 TER119陽性細胞は、チューブの側面に付着する。アタッチされていないTER119負の赤芽球を懸濁液のままになります。 </li><li>ボトムチューブ新ラウンド5ミリリットルのポリプロピレンに細胞懸濁液を注ぐ。 </li><li><html>繰り返し、残留TER119陽性細胞を除去するために3.2.6と3.2.7を繰り返します。 </li><li> 5分間800×gで細胞をペレット化。上清を吸引除去する。 10％FBSを含む1mlのPBSで細胞を再懸濁し、トリパンブルーで生細胞を数える。 </li></ol></li></ol>ウイルスコードするcDNAまたはshRNAで4形質導入<ol><li> 3-5の精製TER119負の胎児肝細胞をプレー×10 5 /ウェルで12ウェルプレートをコーティングしたフィブロネクチン（異なるプレートを用いた場合に細胞数を調整する）である。 </li><li>ウイルス形質導入を容易にするためには10μg/ mlの最終濃度にポリブレンを加える。スピンは、37℃で1〜1.5時間、800×gで、cDNAまたはshRNAをコードするレンチウイルスまたはレトロウイルスで細胞を感染させる。 </li></ol>形質導入TER119陰性マウス胎児肝臓赤芽球の5。文化<ol><li>端末赤血球生成の初期段階での遺伝子の機能をテストするには（ 図1破線）。&quot;&gt; </ol.><ol><li><ol. li="">形質導入された遺伝子および前駆状態の維持の発現を可能にする12時間培養エポ培地で形質導入した細胞（SCF培地）。 </ol.></li><li> 5分間800×gで細胞を遠心。上清を吸引。 12ウェルプレートの各ウェルに培地を含有する1ミリリットルのEpo中で細胞を再懸濁する。 </li><li> 24または48時間培養後、さらなるアッセイのための細胞を採取する。 </li></ol></li><li>端末赤血球生成の後期段階でのテスト遺伝子機能（ 図1の実線）。 <ol><li>文化エポ含む培地中で、すぐに導入された細胞。 </li><li> 24または48時間培養後、さらなるアッセイのための細胞を採取する。 </li></ol></li></ol>フローサイトメトリー分析のために培養された胎児肝細胞の染色6。 <ol><li> 1×PBS中で細胞を洗浄し、再懸濁します。収穫フローサイトメトリー分析のために2×10 5個の培養細胞。 </li><li>培養する暗所で室温で20分間フルオロフォア結合抗体を有する細胞。分化をテストするには、APC結合抗CD71およびPE結合抗TER119を使用しています。摘出をテストするには、他の抗体に加えて、ヘキスト33342染色を使用しています。 </li><li> 1×PBSで細胞を洗浄する。死細胞を染色し、分析から除外するために、1％FBSおよびヨウ化プロピジウム（PI）（1μg/ ml）を含む0.5 mlのPBS中に細胞を再懸濁。 </li><li>流れcytrometric分析を実施する。フローサイトメーターの調整と補償のために最初に単一の色に染色し、染色していない対照細胞を分析します。 </li></ol>

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著者は、彼らが競合する金融利害がないことを宣言します。

ここでは、時系列的に、マウス胎児肝臓端末赤血球生成を分析する独自のシステムを提示する。異なる培養条件の適用を通じて、首尾よく初期および後期段階での端末赤血球生成を解剖した。これは、複数の機能を有する遺伝子のメカニズムを決定するために特に重要である。例えば、RacのGTPアーゼは、端末赤血球生成の各段階において重要な役割を果たしている。端末赤血球生成に影響を?...

赤血球生成は、赤血球を成熟多能造血幹細胞の分化の過程である。この段階的なプロセスは、コミットされた赤血球バースト形成単位（BFU-ES）、急速に分裂赤血球コロニー形成単位（CFU-ES）、及び形態学的に認識可能な赤芽球1,2の形成を含む。 CFU-Eの前駆細胞からの端末赤血球生成は、シーケンシャルエリスロポエチン依存性および非依存性のステージ2,3を伴う。端末赤血球形成の初期段階では、エリスロポエチン（EPO）は、細胞表面上の受容体に結合し、細胞アポトーシスを防止し、迅速な細胞分裂および遺伝子発現1,4を促進する下流のシグナル伝達経路のシリーズを誘導する。端末赤血球生成の後期段階では、赤芽球は、高度に凝縮した核5の端子分裂停止、クロマチンおよび核の凝縮、および押出を受ける。 端末の理解赤血球生成が大幅にin vitroおよびin vivoで 6-9マウスモデルにおけるいくつかの使用の成功によるところが大きい、これ過去数十年で改善されています。これらのモデルの中でも、マウス胎児肝芽球のインビトロ培養中の細胞の精製、速い増殖および分化の容易さを含む多くの利点を提供し、人間の赤血球生成10,11に近い模倣。このシステムでは、マウス胎児肝臓からの赤血球前駆細胞の多数が容易にTER119の単一ステップ精製（成熟赤血球系細胞のマーカー）は、負の赤芽球を単離することができる。赤芽球の二日間の培養の間、これらの細胞の分化は、トランスフェリン受容体（CD71）の表面発現およびTER119抗原12に基づいて、フローサイトメトリー分析によってモニターすることができる。また、末端分化赤芽球の除核は、DNAメーカー（ヘキスト33342）によって検出することができる13。さらに、精製された前駆細胞は、遺伝的に赤血球生成11,13,14上の遺伝子発現の機能の機構研究を容易にする、目的の遺伝子のcDNAまたは小ヘアピンRNA（shRNAは）の外因性発現によって改変することができる。 それは、端末赤血球形成の異なる段階での遺伝子機能を特徴付けることは困難であるので、一方で、高速細胞増殖速度は、両刃の剣であることができる。ほとんどの場合、それは、cDNAまたはshRNAが発現した時点で以降の端末赤血球形成の初期段階における特定の遺伝子の機能は、細胞が既に初期段階に合格したかどうかを決定することは困難である。この問題を解決するために、私たちは、端末赤血球生成の初期および後期段階を詳細に分析するために独自のシステムを開発しました。端末赤血球形成の初期段階では、遺伝的に改変されTER119負赤芽球は、Epoを含まない培地中で培養したが、幹細胞因子（SCF）、IL-6およびFLT3を含むリガンドは、それらの前駆状態を維持し、数13に形質導入されたcDNAまたはshRNAを可能にする。細胞は、細胞の増殖および分化を誘導するために12時間後にEpoを含む培地に変更した。細胞が分化し始めたときにこのように、形質導入されたcDNAまたはshRNAは、既に発現した。端末赤血球生成の後期段階では、TER119負の赤芽球は、すぐ​​に導入後エポ含む培地で培養した。従って、一つの端末赤血球形成の後期段階において、目的の遺伝子の機能を分析することができる。要約すると、本システムの広範な適用は、端末赤血球形成の異なる段階での遺伝子機能を分析に役立つだろう。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Iscove&#39;s Modified Dulbecco&#39;s MediumIMDM (IMDM)</td>
			<td>Gibco</td>
			<td>12440-053</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>GEMINI Bio-product</td>
			<td>700-102P</td>
			<td></td>
		</tr>
		<tr>
			<td>b-mecaptoethanol</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td>0.1 M in IMDM,&#160; 4&#8451;stock</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin solution</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td>100 X</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Hyclone</td>
			<td>SH30034.01</td>
			<td>200 mM</td>
		</tr>
		<tr>
			<td>Stem cell factor (SCF)</td>
			<td>STEMCELL TECH</td>
			<td>2630</td>
			<td>100 ng/ul, -20&#8451; stock</td>
		</tr>
		<tr>
			<td>Mouse FIT3 Ligand (Flt-3L)</td>
			<td>BD Biosciences&#160;</td>
			<td>14-8001-80</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Recombinant Interleukin-6 (IL-6)&#160;</td>
			<td>GEMINI Bio-product</td>
			<td>300-327P</td>
			<td></td>
		</tr>
		<tr>
			<td>fibronectin</td>
			<td>BD Biosciences&#160;</td>
			<td>354008</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>STEMCELL TECH</td>
			<td>9300</td>
			<td>10% stock in IMDM</td>
		</tr>
		<tr>
			<td>Insulin solution, human</td>
			<td>Sigma</td>
			<td>I9278</td>
			<td>4&#8451; stock</td>
		</tr>
		<tr>
			<td>holo-transferrin human</td>
			<td>Sigma</td>
			<td>T0665</td>
			<td>50 mg/ml in Water -20&#8451; stock</td>
		</tr>
		<tr>
			<td>Erythropoietin(Epo)</td>
			<td>PROCRIT</td>
			<td>NOC 59676-303-00</td>
			<td>3,000 U/ml stock</td>
		</tr>
		<tr>
			<td>Streptavidin particles Plus</td>
			<td>BD Pharmingen</td>
			<td>557812</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Magnet</td>
			<td>STEMCELL TECH</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene Round-Bottom Tube, 5 ml</td>
			<td>FALCON</td>
			<td>352063</td>
			<td></td>
		</tr>
	</tbody>
</table>

mouse fetal liver culture system to dissect target gene functions at the early and late stages of terminal erythropoiesis

赤血球生成が急速に形成単位赤血球コロニー（CFU-ES）を分周して、その後、コミット赤血球バースト形成単位（BFU-ES）で始まる動的なプロセスを伴う。 CFU-ESの後、細胞を形態学的に認識され、一般的には、端末赤芽球と呼ばれる。端末赤血球生成の研究のための課題の一つは、時系列的に遺伝子の機能を分析するための実験的アプローチの欠如である。このプロトコルでは、端末赤血球形成の初期及び後期段階での遺伝子機能を決定するためのユニークな戦略を記載している。このシステムでは、マウス胎児肝臓TER119（成熟赤血球細胞マーカー）負の赤芽球を精製し、目的の遺伝子のcDNAや小さなヘアピンRNA（shRNAを）の外因性発現で形質導入。細胞は、外因性cDNAまたはshRNAを可能にしながら、12時間それらの前駆段階を維持するためにエリスロポエチン（EPO）以外の成長因子を含む培地で培養し、その後発現させた。外因性遺伝物質がすでに発現させながら、細胞は、細胞分化および増殖を誘導するために12時間後のEpo培地に変更した。このプロトコルは、端末赤血球生成の初期段階で遺伝子機能の解析を容易にします。後期端末赤血球形成を研究するために、細胞を直ちに形質導入​​後のEpo培地中で培養した。このように、細胞は、既に形質導入​​された遺伝物質を発現した端末赤血球形成の後期に分化させた。私たちは、端末赤血球生成のさまざまな段階での詳細な遺伝子機能を理解する助けとなるこの戦略の一般的な適用を推奨します。

図1は、実験的な戦略の概要を説明します。プロトコルは、端末赤血球生成の初期および後期段階でのシグナル伝達分子の機能を標的とするための2つの独立した条件で構成されています。 TER119陰性胎児肝芽球は、E13.5マウス胎児から精製した。胎児肝臓赤血球系細胞の前および精製後のフローサイトメトリー分析により、精製が（ 図2Aおよび2B）効率的?...

Watch this Scientific Journal Video about Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis at JoVE.com

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

私たちは、端末赤血球生成の初期および後期段階を解剖インビトロマウス胎児肝臓赤芽球培養系を提示。このシステムは、異なる発生段階における特定の遺伝子の機能解析を容易にします。

マウス胎児肝臓培養システムは、ターミナル赤血球の初期および後期段階での標的遺伝子の機能を分析する

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

mouse-fetal-liver-culture-system-to-dissect-target-gene-functions-at

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Immunology and Infection

Unit 1

A Hierarchical Stem-Cell System: Blood Cell Formation

SCIENTISTS IN ACTION

14.8K ビュー.  Northwestern University. 私たちは、端末赤血球生成の初期および後期段階を解剖インビトロマウス胎児肝臓赤芽球培養系を提示。このシステムは、異なる発生段階における特定の遺伝子の機能解析を容易にします。 

ビデオ: Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

細菌付着の研究ではせん断応力を導入する

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

免疫・感染学

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

幹細胞ベースの遺伝子治療腫瘍モデルとしてBLTヒト化マウスを用いた

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target.
The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design.
Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for lead discovery. The following article highlights how the Seattle Structural Genomics Center for Infectious Disease utilizes a multi-target approach for gene-to-structure determination of the PB2 influenza A subunit.

インフルエンザポリメラーゼPB2サブユニットの遺伝子の構造決定のためのマルチターゲット並列処理アプローチ

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States ...

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

遺伝子発現のすべての段階でウイルス機能を定量化するためのワクレポーターウイルス

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

母体·胎児界面におけるマウス組織からの白血球の分離

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

母体・胎児のインターフェイスでの感染症を研究するためにヒト胎盤および脱落膜器官培養

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Flow Cytometry-based Assay for the Monitoring of NK Cell Functions

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and virus-infected or malignantly transformed cells due to a set of germline encoded inhibitory and activating receptors. Upon virus or tumor cell recognition a variety of different NK cell functions are initiated including cytotoxicity against the target cell as well as cytokine and chemokine production leading to the activation of other immune cells. It has been demonstrated that accurate NK cell functions are crucial for the treatment outcome of different virus infections and malignant diseases. Here a simple and reliable method is described to analyze different NK cell functions using a flow cytometry-based assay. NK cell functions can be evaluated not only for the whole NK cell population, but also for different NK cell subsets. This technique enables scientists to easily study NK cell functions in healthy donors or patients in order to reveal their impact on different malignancies and to further discover new therapeutic strategies.

A simple and reliable method is described here to analyze a set of NK cell functions such as degranulation, cytokine and chemokine production within different NK cell subsets.

NK細胞の機能を監視するためのサイトメトリーに基づくアッセイを流します

Natural killer (NK) cells are an important part of the human tumor immune surveillance system. NK cells are able to distinguish between healthy and ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

複製サイクルの初期段階を監視するためにB型肝炎ウイルスのレポーターシステムの開発

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

エスケープ中和抗体でインフルエンザ ウイルスの亜種の世代

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

インフルエンザ血球凝集素抗体による Fc を介したエフェクター機能を評価する方法

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...