CKは、コロンビア大学大学院のプログラムによってサポートされています。 UBは、アメリカの白血病とリンパ腫協会、白血病研究基金からの新しい研究者賞の受信者のフェローであり、コロンビア大学新学部スタートアップ資金によってサポートされています。

1。脾臓B -リンパ球の単離と刺激<ol><li>年齢の2〜3ヶ月の間にAID欠損マウスから脾臓を12を回収する。脾臓の単離は無菌条件下で行われ、臓器が一時的に15パーセントウシ胎児血清（FBS）を含む冷リン酸緩衝生理食塩水（PBS）に保存されています。 </li><li>脾臓を無菌組織培養フードに移し、2.5％FBSを添加したPBS 5mLにホモジナイズする。 15 mLのファルコンチューブにホモジネートを移す。 </li><li> 4℃で10分間1000rpmでホモジナイズした脾臓組織のサンプルを遠心° Cの細胞をペレット化する。 </li><li>遠心分離後の上清をデカント。赤血球の混入を避けるために、10 mLの赤血球の細胞ペレット（RBC）、7分間室温で溶解バッファーを再懸濁します。 </li><li>遠心分離機は、10分間1000rpmでサンプルし、上清をデカントし、2.5％FBSを含有する10mLのPBSで細胞を懸濁します。 </li><li> 100μL分注しを取り出して、死細胞を除外するためにトリパンブルーを使用して、標準血球計を使用して1:1000希釈で細胞をカウント。約20〜30万個の細胞は、脾臓当たり得ることができます。 </li><li> 10分間1000rpmでサンプルを遠心し、上清をデカントする。 </li><li> 0.5パーセント90μLあたり10 7個の細胞の濃度でBSA / PBSを含む2mMのEDTAでペレットを再懸濁する。 </li><li>特に非B細胞をバインドするために細胞に抗CD43抗体被覆磁性ビーズを追加。 90μLあたり10μLビーズ（10 7細胞）を使用してください。よく混和して、4℃でインキュベート℃で20分間。 </li><li>細胞が標識されていると、チューブの上部には、2.5％FBS / PBSを添加し、それらを洗う。 10分間、1000rpmでサンプルを遠心分離します。 </li><li>上清をデカント。 mlあたり10 × 10 7細胞の濃度で0.1％BSA / PBSで細胞を再懸濁します。 </li><li>マグネット分離装置に磁気ソート列をマウントします。直接フロースルーを収集し、プライムと洗浄する3ML 0.5％BSA / PBSでカラムをロードする列の下に位置コニカルチューブを。 </li><li>洗浄流体が流れると、新しいコレクションチューブに交換してください。サンプルは1 mLの未満の場合、0.5％BSA / PBS / EDTAで1 mLにサンプルの音量を調整し、カラムにサンプルをロードする。列あたりのセル数だけを1 mLの最大値をロードし、必要に応じて複数の列を使用してください。 </li><li>細胞が受動的にカラムに流すと列の下のコニカルチューブに未結合の細胞を収集することができます。スルーフロー収集を継続しながら、3 mLの0.5％BSA / PBSでカラムを4回洗浄する。 </li><li>すべてのCD43陰性休止B細胞を濃縮されるのフロースルーを収集する。列を捨てる。フロースルー、1000rpmで10分間の遠心分離します。 </li><li>上清を排出し、10 mLの培地（15％FCS / +グルタミン、ペン/球菌、非必須アミノ酸、50μMΒ- ME）を追加する</li><li> mlあたり10 6細胞での細胞と文化を数える。 </li><li> B細胞の成長および増殖を刺激するためには、それぞれ、最終濃度が20μg/ mLの、1μg/ mLとなるような組換えIL - 4と抗CD40抗体を用いて培地を補足し、培養フラスコに細胞を移す。 </li><li>一晩培養細胞を。細胞をカウントし、必要に応じてIL - 4とCD40を追加し、mlあたり10 6個の細胞に希釈する。 </li><li>ウイルス感染前に72時間の培養細胞。 </li></ol> 2。プロデューサー細胞のトランスフェクションカチオン性脂質またはリン酸カルシウムのいずれかを使用してトランスフェクションするための確立されたプロトコルを使用してください。プレート当たり100μgのプラスミドDNAを用いたDNA構築物ごとにトランスフェ10cmの3プレート、。我々は定期的にリン酸カルシウムトランスフェクション法13、14を使用し、クロロキンを添加したときに高いウイルス力価に注意してください。 <ol><li>トランスフェクトされるプロデューサー細胞のような60％-70％コンフルエントフェニックスの細胞を使用してください。この合流の細胞数で10cmのプレートあたり5万セルでなければなりません。 </li><li>トランスフェクションの1時間前には、フェニックスの細胞増殖のための新鮮な完全培地と培地を交換してください。 </li><li>滅菌eppendorpfチューブで、、包装構造のDNAの100μgを組み合わせる0.5M CaCl 2を250μL、および滅菌水を用いて500μLに最終的な音量を調節</li><li> 1 mLのピペットを使用して、精力的に15mlのポリプロピレンチューブに500μL2X HNPでこのソリューションを混在させる</li><li>この混合物が8〜10分間室温で休ませることができます。この時間の間に沈殿物が形成されます。 </li><li>トランスフェクション混合物を均一に全体に混合物を配布する媒体を旋回しながら細胞に滴下を追加。 </li><li> 37細胞をインキュベート℃で12時間を。 </li><li> 12時間トランスフェクション後は、8ミリリットル完全なB細胞の増殖培地で培地を交換し、37℃のインキュベーターにセルを返します。 </li></ol> 3。刺激脾臓B細胞の感染<ol><li>ポストtransfe 48hrフェニックスの細胞から上清を除去ctionとあらゆる細胞破片を除去するために0.2ミクロンフィルターを介して上清を渡します。 </li><li>プレ活性化B細胞（ステップ1のように）3mlでウイルスを含む上清を3 mLを混ぜる。ウイルスの細胞への吸着性を高めるために、液あたり16μgの最終濃度でポリブレンを追加。 </li><li> 30℃で90分間2500rpmで遠心して細胞に感染℃に</li><li> 37℃でさらに48〜72時間インキュベートスピン、° Cは、細胞の成長と増殖を可能にした直後に。 </li></ol> 4。フローサイトメトリー分析<ol><li>フローサイトメーターを使用して、B220と表面IgG1の表面発現のため細胞を分析する。細胞の表面でのIgG1の存在は、クラススイッチ組み換えがAID遺伝子のレトロウイルス相補経由して成功した救助の結果として発生したことを示します。 </li></ol> 5。代表的な結果私たちは正常（AID - / - ）AID欠損からタンパク質因子の活性化誘導シチジンデアミナーゼ（AID）の欠乏を救出しているレトロウイルス導入を用いてB -リンパ球。簡単に言えば、我々は、PMX - MAID - GFP 15から18でフェニックス細胞をトランスフェクトすることにより、マウスのAID（MAID）タンパク質を発現する組換えレトロウイルスを生成。収穫されたウイルスは、 生体外でのクラススイッチ組み換え（CSR）刺激の条件19でAID欠損マウスから得られたAID欠損B細胞に感染させた。感染したB細胞は、フローサイトメトリー分析を用いて培養中の72時間後にCSRへのIgG1のために分析した。図2は、CSRは、AIDの発現の救助の結果として発生したことを示す、AIDによるB細胞の表面におけるIgG1の検出ではなく、空の構造を示しています。 <img src="/files/ftp_upload/2371/2371fig1.jpg" alt="図1" /> 図1：プロデューサー細胞内でレト ​​ロウイルスパッケージングのスキーム：以前は15を説明するようにΨで表される目的の遺伝子が、、、PMX - IRES - GFPへサブクローニングした。プラスミドは、リン酸カルシウム法13、14を使用して、ギャグ、polおよびエンベロープタンパク質（フェニックス、Orbigen）を作り出すことができるエコトロピックウイルスパッケージング細胞株にトランスフェクトした。トランスフェクション48時間後、上清を含有するウイルス粒子は、マウスから得られたターゲットのプライマリのB細胞への感染のために採取した。 <img src="/files/ftp_upload/2371/2371fig2.jpg" alt="図2" /> 図2：AID欠損B細胞は、抗CD40で刺激し、IL - 4は、PMX - MAID - GFPまたは単独でGFPを発現するレトロウイルスを含むレトロウイルスを感染させた。 IgG1のへのCSRのレベルは、FACS分析により評価した。

<ol>
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利害の衝突は宣言されません。

ここで説明すると、 図1に示すように、脾臓B細胞のレトロウイルス導入はlymphopoesisの発達のイベントの多くは転写制御1、2によって制御されるため、B -リンパ球の研究に有用な遺伝学的ア ​​プローチです。 B細胞の成熟の後の段階では、CD40Lを介してトリガするB細胞増殖、細胞周期への参入、および増殖11、20の誘導に必須である。 図2に示すように...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>FCS</td>
<td>&nbsp;</td>
<td>Atlanta Biologicals</td>
<td>S11550</td>
<td>&nbsp;</td>
<tr>
<td>RPMI</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>22400</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Invitrogen/Gibco</td>
<td>20012</td>
<td>&nbsp;</td>
<tr>
<td>Red Blood Cell (RBC) lysis buffer</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>R7757</td>
<td>&nbsp;</td>
<tr>
<td>CD43 beads</td>
<td>&nbsp;</td>
<td>Miltenyi</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>B Cell Complete Media</td>
<td>&nbsp;</td>
<td>Various components</td>
<td>Various Components</td>
<td>RPMI, 15% FCS, 1% Non-Essential Amino Acids, 1% Sodium Pyruvate, 1% HEPES, 1% Pen-Strep, 50&mu;M &beta;-Mercaptoethanol</td>
</tr>
<tr>
<td>IL-4</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Anti-CD40</td>
<td>&nbsp;</td>
<td>BD Pharmigen</td>
<td>553787</td>
<td>&nbsp;</td>
<tr>
<td>polybrene</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>107689 &#x2028;</td>
<td>&nbsp;</td>
<tr>
<td>Chloroquine diphosphate salt</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>C6628</td>
<td>Used at 100mM</td>
</tr>
<tr>
<td>Phoenix Eco cells (Murine)</td>
<td>&nbsp;</td>
<td>Orbigen</td>
<td>RVC-10002</td>
<td>&nbsp;</td>
<tr>
<td>PE-Cy5-&alpha;-mouse-CD45R (B220)</td>
<td>&nbsp;</td>
<td>eBioscience</td>
<td>15-0452-81</td>
<td>&nbsp;</td>
<tr>
<td>PE-&alpha;-mouse-IgG1</td>
<td>&nbsp;</td>
<td>BD Pharmigen</td>
<td>A85-1</td>
<td>&nbsp;</td>		</tbody>
		</table>
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recombinant retroviral production and infection of b cells

真核細胞における遺伝子のトランスジェニック発現は、目的の遺伝子が生じる表現型の解析を容易にするために異種発現系の制御下で発現されている強力な逆遺伝学的アプローチです。このアプローチは、遺伝子産物の変異型を表現するために、または遺伝子産物のドミナントネガティブ型を過剰発現するように、通常の生物で発見されていない遺伝子を発現させることができる。それは転写調節が2-4でレビューB細胞1の分化における主要な制御機構である造血系の研究では特に便利です。 マウス遺伝学は、人間の遺伝子と疾病の研究のための強力なツールです。マウスとヒトのゲノムの比較解析は、ゲノム5の90％以上のシンテニーの保全を明らかにする。また、多くのマウスモデルで使用されている技術のは、例えば、遺伝子の混乱と対立遺伝子交換6ヒト遺伝子の研究にも適用可能であるただし 、トランスジェニックマウスの作成 ​​は、財政的、技術的性質の両方のリソースが大量に必要になります。いくつかのプロジェクトでは、ノックアウトマウス系統（KOMP、EUCOMM、NorCOMM）または大規模な努力とコラボレーション7を必要とする特異的変異誘発株（理研）、のライブラリをコンパイルし始めている。したがって、それは最初のマウスモデルに進行する前に、初代培養細胞の細胞培養モデルにおける所望の遺伝子の表現型を検討することが望ましい。 レトロウイルスDNAは安定した結果、好ましくは転写単位またはCpGアイランド内、または近くに、宿主のDNAに統合されており、転写サイレンシング8 9を避けながら、関心のパッケージ化遺伝子の発現を遺伝。遺伝子は、転写とタンパク質生産の高効率で、その結果、高効率レトロウイルスプロモーターの制御下に転写されます。このため、レトロウイルス発現はトランスフェクションが困難な細胞に使用することができる、細胞が有糸分裂中にアクティブな状態で提供される。ウイルスの構造遺伝子をパッケージング細胞株内に含まれているため、目的の遺伝子をクローニングするために用いられる発現ベクターは、両方のウイルスの復帰の可能性を排除し、ウイルス上清での作業の安全性を高めるウイルス、のない構造遺伝子が含まれていないは感染性ビリオンは10を生成されませんように。 ここでは、組換えレトロウイルスの生産と脾臓B細胞のその後の感染のためのプロトコルを提示する。単離後、培養脾臓細胞はB細胞の増殖と分化11のバーストを誘導するのTh由来のリンホカイ ​​ンと抗CD40で刺激される。このプロトコルは、B細胞は形質細胞分化を誘導するが、前の抗原刺激に対する初期造血のイベントに続いて脾臓から分離されているとして、後期B細胞の発達と分化に発生するイベントの研究に最適です。

Watch this Scientific Journal Video about Recombinant Retroviral Production and Infection of B Cells at JoVE.com

Recombinant Retroviral Production and Infection of B Cells

の遺伝子の構造と機能解析の効率的なシステム<em&gt;生体外で</em脾臓B -リンパ球の&gt;文化が説明されています。このメソッドは、ヘルパーフリー、ecotrophicパッケージング細胞株における組換えレトロウイルスの生産の利点を取ります。安定した、クラススイッチ組み換えを受けたB細胞上の表面の抗体の産生につながる達成される一次リンパ球内で目的遺伝子の発現を遺伝。

B細胞の組換えレトロウイルスの生産と感染症

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

recombinant-retroviral-production-and-infection-of-b-cells

Research

JoVE Journal

Immunology and Infection

14.0K ビュー.  Columbia University College of Physicians and Surgeons. の遺伝子の構造と機能解析の効率的なシステム生体外で文化が説明されています。このメソッドは、ヘルパーフリー、ecotrophicパッケージング細胞株における組換えレトロウイルスの生産の利点を取ります。安定した、クラススイッチ組み換えを受けたB細胞上の表面の抗体の産生につながる達成される一次リンパ球内で目的遺...

ビデオ: Recombinant Retroviral Production and Infection of B Cells

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

B細胞のCD40 -活性化のモデルマウス

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

免疫・感染学

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

マウスT細胞におけるT細胞受容体のレトロウイルス導入

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

組換えアデノ随伴ウイルスベクターの産生と力価測定

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

アデノウイルス由来のシステムの助けを借りて効率的な組換えパルボウイルスの生産

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

真のIgE発現するマウスB系統細胞の検出

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

不死化ソートB細胞からの組換えヒトIgGモノクローナル抗体の作製

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

骨髄前駆細胞のレトロウイルス形質導入は、T細胞受容体Retrogenicマウスを生成するために、

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

組換えタンパク質の部位特異的糖体外のシステインのチオール基を対象と

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

&quot;Liver-on-a-Chip&quot; Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

初代肝細胞とクッパー細胞 B 型肝炎ウイルス感染の「肝臓-オン-チップ」文化

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

マウスB-1a細胞におけるCXCR4のレトロウイルス過剰発現と骨髄およびIgM産生への標的B-1a細胞の移行のための養子転移

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...