pMSCV - AID - I - Thy1.1とpKat2ベクトルはDGシャッツから親切な贈り物だったとpVSV - G、pRSV - REV、およびpMDLg / pRREベクトルはBRカレンから親切な贈り物でした。

<ol>
	<li>Di Noia, J. M., Neuberger, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+antibody+somatic+hypermutation.">Molecular mechanisms of antibody somatic hypermutation.</a> Annu. Rev. Biochem. 76, 1-22 (2007).</li><li>Peled, J. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biochemistry+of+somatic+hypermutation.">The biochemistry of somatic hypermutation.</a> Annu. Rev. Immunol. 26, 481-511 (2008).</li><li>Longerich, S., Basu, U., Alt, F., Storb, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AID+in+somatic+hypermutation+and+class+switch+recombination.">AID in somatic hypermutation and class switch recombination.</a> Curr. Opin. Immunol. 18, 164-174 (2006).</li><li>Bardwell, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+somatic+hypermutation+and+reduced+class-switch+recombination+in+exonuclease+1-mutant+mice.">Altered somatic hypermutation and reduced class-switch recombination in exonuclease 1-mutant mice.</a> Nat. Immunol. 5, 224-229 (2004).</li><li>Martomo, S. A., Yang, W. W., Gearhart, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+role+for+Msh6+but+not+Msh3+in+somatic+hypermutation+and+class+switch+recombination.">A role for Msh6 but not Msh3 in somatic hypermutation and class switch recombination.</a> J. Exp. Med. 200, 61-68 (2004).</li><li>Phung, Q. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+hypermutation+at+G+and+C+nucleotides+in+immunoglobulin+variable+genes+from+mice+deficient+in+the+MSH2+mismatch+repair+protein.">Increased hypermutation at G and C nucleotides in immunoglobulin variable genes from mice deficient in the MSH2 mismatch repair protein.</a> J. Exp. Med. 187, 1745-1751 (1998).</li><li>Rada, C., Ehrenstein, M. R., Neuberger, M. S., Milstein, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hot+spot+focusing+of+somatic+hypermutation+in+MSH2-deficient+mice+suggests+two+stages+of+mutational+targeting.">Hot spot focusing of somatic hypermutation in MSH2-deficient mice suggests two stages of mutational targeting.</a> Immunity. 9, 135-141 (1998).</li><li>Rada, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoglobulin+isotype+switching+is+inhibited+and+somatic+hypermutation+perturbed+in+UNG-deficient+mice.">Immunoglobulin isotype switching is inhibited and somatic hypermutation perturbed in UNG-deficient mice.</a> Curr. Biol. 12, 1748-1755 (2002).</li><li>Delbos, F., Aoufouchi, S., Faili, A., Weill, J. C., Reynaud, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+polymerase+eta+is+the+sole+contributor+of+A/T+modifications+during+immunoglobulin+gene+hypermutation+in+the+mouse.">DNA polymerase eta is the sole contributor of A/T modifications during immunoglobulin gene hypermutation in the mouse.</a> J. Exp. Med. 204, 17-23 (2007).</li><li>Masuda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+polymerase+eta+is+a+limiting+factor+for+A:T+mutations+in+Ig+genes+and+contributes+to+antibody+affinity+maturation.">DNA polymerase eta is a limiting factor for A:T mutations in Ig genes and contributes to antibody affinity maturation.</a> Eur. J. Immunol. 38, 2796-2805 (2008).</li><li>Lim, D. S., Hasty, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+in+mouse+rad51+results+in+an+early+embryonic+lethal+that+is+suppressed+by+a+mutation+in+p53.">A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53.</a> Mol. Cell. Biol. 16, 7133-7143 (1996).</li><li>Xiao, Y., Weaver, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+gene+targeted+deletion+by+Cre+recombinase+demonstrates+the+requirement+for+the+double-strand+break+repair+Mre11+protein+in+murine+embryonic+stem+cells.">Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells.</a> Nucleic. Acids. Res. 25, 2985-2991 (1997).</li><li>Zhu, J., Petersen, S., Tessarollo, L., Nussenzweig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+the+Nijmegen+breakage+syndrome+gene+NBS1+leads+to+early+embryonic+lethality+in+mice.">Targeted disruption of the Nijmegen breakage syndrome gene NBS1 leads to early embryonic lethality in mice.</a> Curr. Biol. 11, 105-109 (2001).</li><li>Luo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+mRad50+causes+embryonic+stem+cell+lethality,+abnormal+embryonic+development,+and+sensitivity+to+ionizing+radiation.">Disruption of mRad50 causes embryonic stem cell lethality, abnormal embryonic development, and sensitivity to ionizing radiation.</a> Proc. Natl. Acad. Sci. U. S. A. 96, 7376-7381 (1999).</li><li>Pavri, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation-induced+cytidine+deaminase+targets+DNA+at+sites+of+RNA+polymerase+II+stalling+by+interaction+with+Spt5.">Activation-induced cytidine deaminase targets DNA at sites of RNA polymerase II stalling by interaction with Spt5.</a> Cell. 143, 122-133 (2010).</li><li>Lee-Theilen, M., Matthews, A. J., Kelly, D., Zheng, S., Chaudhuri, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CtIP+promotes+microhomology-mediated+alternative+end+joining+during+class-switch+recombination.">CtIP promotes microhomology-mediated alternative end joining during class-switch recombination.</a> Nat. Struct. Mol. Biol. 18, 75-79 (2011).</li><li>Basu, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA+exosome+targets+the+AID+cytidine+deaminase+to+both+strands+of+transcribed+duplex+DNA+substrates.">The RNA exosome targets the AID cytidine deaminase to both strands of transcribed duplex DNA substrates.</a> Cell. 144, 353-363 (2011).</li><li>Yabuki, M., Fujii, M. M., Maizels, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MRE11-RAD50-NBS1+complex+accelerates+somatic+hypermutation+and+gene+conversion+of+immunoglobulin+variable+regions.">The MRE11-RAD50-NBS1 complex accelerates somatic hypermutation and gene conversion of immunoglobulin variable regions.</a> Nat. Immunol. 6, 730-736 (2005).</li><li>Sale, J. E., Neuberger, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TdT-accessible+breaks+are+scattered+over+the+immunoglobulin+V+domain+in+a+constitutively+hypermutating+B+cell+line.">TdT-accessible breaks are scattered over the immunoglobulin V domain in a constitutively hypermutating B cell line.</a> Immunity. 9, 859-869 (1998).</li><li>Cumbers, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+iterative+affinity+maturation+of+antibodies+in+vitro+using+hypermutating+B-cell+lines.">Generation and iterative affinity maturation of antibodies in vitro using hypermutating B-cell lines.</a> Nat. Biotechnol. 20, 1129-1134 (2002).</li><li>Papavasiliou, F. N., Schatz, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-cycle-regulated+DNA+double-stranded+breaks+in+somatic+hypermutation+of+immunoglobulin+genes.">Cell-cycle-regulated DNA double-stranded breaks in somatic hypermutation of immunoglobulin genes.</a> Nature. 408, 216-221 (2000).</li><li>Parsa, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AID+mutates+a+non-immunoglobulin+transgene+independent+of+chromosomal+position.">AID mutates a non-immunoglobulin transgene independent of chromosomal position.</a> Mol. Immunol. 44, 567-575 (2007).</li><li>Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+instability+of+V+region+hypermutation+in+the+Ramos+Burkitt's+lymphoma+cell+line.">Clonal instability of V region hypermutation in the Ramos Burkitt's lymphoma cell line.</a> Int. Immunol. 13, 1175-1184 (2001).</li><li>Ukai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+a:T+mutations+is+dependent+on+cellular+environment+but+independent+of+mutation+frequency+and+target+gene+location.">Induction of a:T mutations is dependent on cellular environment but independent of mutation frequency and target gene location.</a> J. Immunol. 181, 7835-7842 (2008).</li><li>Dull, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+third-generation+lentivirus+vector+with+a+conditional+packaging+system.">A third-generation lentivirus vector with a conditional packaging system.</a> J. Virol. 72, 8463-8471 (1998).</li><li>Nakamura, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+frequency+class+switching+of+an+IgM++B+lymphoma+clone+CH12F3+to+IgA++cells.">High frequency class switching of an IgM+ B lymphoma clone CH12F3 to IgA+ cells.</a> Int. Immunol. 8, 193-201 (1996).</li><li>Muramatsu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+expression+of+activation-induced+cytidine+deaminase+(AID),+a+novel+member+of+the+RNA-editing+deaminase+family+in+germinal+center+B+cells.">Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells.</a> J. Biol. Chem. 274, 18470-18476 (1999).</li></ol>

利害の衝突は宣言されません。

前述したように、抗体の多様化のための細胞株のモデルは、抗体の多様化中に別のステップに影響を与える新規タンパク質を同定するために人気の出発点となっている。我々はここにラモスB細胞株におけるノックダウンまたは過剰発現するタンパク質のいずれかにウイルス感染を使用するための方法を提示し、SHMの影響を調べる。 これらの研究では、WTラモスとAID ?...

推奨試薬-これらのほとんどは他のベンダーから同様の製品で置換されていてもよい。 <table border="1" ><tbody><tr><th>試薬の名前</th><th >会社</th><th>カタログ番号</th><th>コメント</th></tr><tr><td > 6ウェル明確なTC処理プレート</td><td >コー​​ニング</td><td> 3516 </td><td ></td></tr><tr><td > 10 mLのBDルアーLoksyringes </td><td > BDメディカル</td><td > 309604 </td><td ></td></tr><tr><td> 24ウェル透明なTC処理プレート</td><td >コー​​ニング</td><td > 3526 </td><td></td></tr><tr><td > 96ウェル透明な平底ポリスチレンTC -処理マイクロプレート</td><td >コー​​ニング</td><td > 3596 </td><td ></td></tr><tr><td > 100ミリメートルTC処理培養皿</td><td >コー​​ニング</td><td > 430167 </td><td ></td></tr><tr><td >アクロディスクシリンジフィルター、孔径0.45μm </td><td >ポールライフサイエンス</td><td > 4604 </td><td ></td></tr><tr><td >寒天</td><td > Teknova </td><td > A7777 </tD&gt; <td ></td></tr><tr><td >アガロース</td><td > GeneMate </td><td> E - 3120 - 500 </td><td ></td></tr><tr><td >アンピシリン</td><td>シグマ</td><td > A0166 </td><td>水中では100 mg / mLの</td></tr><tr><td > BD FACSCanto IIフローサイトメーター</td><td> BDバイオサイエンス</td><td ></td><td >または類似の</td></tr><tr><td> BDファルコン丸底のポリスチレンチューブ</td><td> BDバイオサイエンス</td><td > 352054 </td><td > FACS用</td></tr><tr><td > BOSC 23細胞</td><td > ATCC </td><td > CRL - 11270 </td><td ></td></tr><tr><td > 37の可能なCO 2インキュベーター° C </td><td ></td><td ></td><td ></td></tr><tr><td > DMEM（ダルベッコ改変イーグル培地） </td><td >シグマ</td><td> D6429 </td><td ></td></tr><tr><td > FBS（ウシ胎児血清） </td><td >ジェミニバイオ製品</td><td > 100から106 </td><td ></td></tr><tr><td > FITCα-ラットCD90/mouse CD90.1抗体</td><td > BioLegend </td><td > 202503 </td><td > FITCα- Thy1.1 </td></tr><tr><td > FuGENE 6トランスフェクション試薬</td><td >ロッシュ</td><td > 11814443001 </td><td></td></tr><tr><td > HEPES緩衝液</td><td >インビトロジェン</td><td > 15630-080 </td><td ></td></tr><tr><td > KAPAハイファイDNAポリメラーゼ</td><td > KAPAバイオシステムズ</td><td > KK2101 </td><td ></td></tr><tr><td > LB培地（溶原スープ - ルリア）パウダー</td><td >ディフコ</td><td > 240230 </td><td ></td></tr><tr><td > MISSION TRC shRNAの細菌のグリセロールストック</td><td >シグマ</td><td ></td><td> shRNAベクター</td></tr><tr><td > NCS（新生児ウシ血清） </td><td >ジェミニバイオ製品</td><td > 100から504 </td><td ></td></tr><tr><td> PBS（リン酸緩衝液） </td><td >インビトロジェン</td><td> 70011 </td><td >水で1Xに希釈</td></tr><tr><td > PEα-ヒトIgM抗体</td><td > BioLegend </td><td > 314508 </td><td ></td></tr><tr><td > PGS（ペニシリン - ストレプトマイシン - グルタミン溶液） </td><td >ジェミニバイオProducts </td><td > 400から110 </td><td ></td></tr><tr><td>ポリブレン（臭化ヘキサジメトリン） </td><td >シグマ</td><td > 107689 </td><td >水に10 mg / mLの</td></tr><tr><td > PureYieldプラスミドミディプレップシステム</td><td >プロメガ</td><td > A2495 </td><td></td></tr><tr><td>ピューロマイシン</td><td>シグマ</td><td > P8833 </td><td >水の250μg/ mLの</td></tr><tr><td > QIAquickゲル抽出キット</td><td > QIAGEN </td><td > 28706 </td><td ></td></tr><tr><td>ラモス（RA 1）細胞</td><td > ATCC </td><td > CRL - 1596 </td><td ></td></tr><tr><td > RPMI - 1640培地</td><td >シグマ</td><td > R8758 </td><td ></td></tr><tr><td >スーパースクリプトII </td><td >インビトロジェン</td><td > 18064-022 </td><td></td></tr><tr><td > SYBR FAST定量PCRキット</td><td > KAPAバイオシステムズ</td><td > KK4601 </td><td ></td></tr><tr><td > Taq DNAポリメラーゼ</td><td >インビトロジェン</td><td > 18038-042 </td><td ></td></tr><tr><td > TOPO TAクローニングキット</td><td >体外ジェン</td><td > K4520 - 01 </td><td ></td></tr><tr><td > TRIZOL </td><td >インビトロジェン</td><td > 15596-026 </td><td ></td></tr><tr><td >ウィザードSVゲノムDNA精製システム</td><td >プロメガ</td><td > A2361 </td><td ></td></tr><tr><td > X - Galを[5 - ブロモ-4 - クロロ-3 - indoyl -β- D - galatopyranoside] </td><td > Growcells </td><td > C - 5687 </td><td > DMSOに40 mg / mLの</td></tr></tbody></table>

assessing somatic hypermutation in ramos b cells after overexpression or knockdown of specific genes

B細胞は、V（D）J組換えにより生成された低親和性の抗体とその生活を始める。しかし、病原体を検出すると、免疫グロブリン（Ig）遺伝子の可変（V）領域は、高親和性抗体の1,2の結果、約10万倍以上の体細胞超変異（SHM）を通じて、ゲノムの他の部分よりも変異している。さらに、クラススイッチ組み換え（CSR）は、特定の病原体に必要とされる免疫応答の種類に応じて、異なるエフェクター機能を有する抗体を産生する。 CSRとSHMの両方は、ウラシルを生成するためにDNAのシトシン残基をdeaminates活性化誘導型シチジンデアミナーゼ（AID）によって開始されています。これらのウラシルは、最終的には突然変異と1-3組換えにつながる、修復経路のエラーが発生しやすいフォームによって処理されます。 マウスでの研究の組み合わせから来るSHMとCSRの分子の詳細、主要な細胞、細胞株、およびセルの- fの私たちの現在の理解REE実験。マウスモデルは、多くの修復因子（例えば雄、MSH2、MSH6、EXO1、およびポリメラーゼη）4-10のために重要な役割を示す遺伝子ノックアウトとゴールドスタンダードのまま。ただし、すべての遺伝子がノックアウトの研究のために適している。例えば、いくつかの二重鎖切断修復タンパク質のノックアウトはembryonically致死的またはB細胞の開発11-14損なう。また、時々SHMやCSRにおけるタンパク質の特定の機能をノックアウトによって引き起こされる多くの世界的な欠陥によってマスクされることがあります。さらに、マウスでの実験以来、細胞株における個々の遺伝子の発現が候補遺伝子15-18識別し、特徴付けるために、ますます人気の第一歩となっている変更、時間がかかる場合があります。 ラモス-構成的にSHMを受けるバーキットリンパ腫細胞株は- SHM 18-24勉強に人気の細胞ラインのモデルとなっています。ラモス細胞の一つの利点は、内蔵の便利なセミを持っていることです。SHMの定量的尺度。野生型の細胞は、突然変異、IgM抗体の発現をノックアウト変異のいくつかをピックアップとして、IgMを発現しており。したがって、蛍光活性化細胞のスキャン（FACS）によってIgMの損失をアッセイすることSHMのレベルのための読み出し迅速に提供します。 SHMのより定量的な測定は、直接抗体遺伝子をシーケンシングすることによって得ることができる。 ラモス細胞はトランスフェクションが困難なので、我々はそれぞれ過剰発現カセットまたは短鎖ヘアピンRNA（shRNA）、のいずれかを含むレトロウイルスやレンチウイルスのコンストラクトで細胞を感染させることによって増加または個々の遺伝子の発現を低下している安定した誘導体を生成する。ここで、我々はラモスの細胞に感染してから、SHM（図1）上の特定の遺伝子の役割を調べるために、これらの細胞を使用する方法について説明します。

Watch this Scientific Journal Video about Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes at JoVE.com

Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

我々は、過剰発現またはshRNAを含む人間ラモスB細胞株とどのようにこれらの細胞で体細胞高頻度変異を測定するためのコンストラクトのレトロウイルスやレンチウイルス感染症を実行する方法について説明します。

特定の遺伝子の過剰発現やノックダウン後にラモスB細胞に体細胞高頻度変異の評価

B cells start their life with low affinity antibodies generated by V(D)J recombination.  However, upon detecting a pathogen, the variable (V) region of an ...

assessing-somatic-hypermutation-ramos-b-cells-after-overexpression-or

Research

JoVE Journal

Immunology and Infection

18.1K ビュー.  Duke University. 我々は、過剰発現またはshRNAを含む人間ラモスB細胞株とどのようにこれらの細胞で体細胞高頻度変異を測定するためのコンストラクトのレトロウイルスやレンチウイルス感染症を実行する方法について説明します。

ビデオ: Assessing Somatic Hypermutation in Ramos B Cells after Overexpression or Knockdown of Specific Genes

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

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

マウスB細胞を精製における誘導とクラススイッチ組み換えの評価

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

同種造血幹細胞移植後のウイルス感染を予防/治療するMultivirus -特異的T細胞の生成

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

細胞運命を以下に<em&gt; E大腸菌の</emラムダファージによる感染後&gt;

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

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 ...

Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate and enrich these rare cells for studies of their phenotype and biology. Approaches are inevitably based on the principle that B cells recognize native antigen by virtue of cell surface receptors that are representative in specificity of antibodies that will eventually be secreted by their differentiated daughters. Perhaps the most obvious approach to the problem involves use of fluorochrome-conjugated antigens in conjunction with fluorescence-activated cell sorting (FACS). However, the utility of these methods is limited by cell frequency and the achievable rate of analysis and isolation by electronic sorting. A novel method to enrich rare antigen-specific B cells using magnetic nanoparticles that results in high yield enrichment of antigen-reactive B cells from large starting cell populations is described. This method enables improved monitoring of the phenotype and biology of antigen reactive cells before and following in vivo antigen encounter, such as after immunization or during development of autoimmunity.

A simple yet effective method that employs magnetic nanoparticles to detect and enrich antigen-reactive B cells for functional and phenotypic analysis is described.

表現型および機能解析のためのレア抗原特異的B細胞の検出および濃縮

B cells reactive with a specific antigen usually occur at a frequency of &lt;0.05% of lymphocytes. For decades researchers have sought methods to isolate ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

ヒトの過剰発現と精製<em&gt;シス</em&gt; - プレニルトランスフェラーゼ<em&gt;大腸菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

まれな抗原特異的 B 細胞血液の循環から弁別に対するモノクローナル抗体の生成

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Assessing Cellular Stress and Inflammation in Discrete Oxytocin-secreting Brain Nuclei in the Neonatal Rat Before and After First Colostrum Feeding

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression of proteins known to respond to metabolic stress was measured in brain-nuclei isolates using Western blotting. This was done to assess whether metabolic stress-induced nutrient insufficiency in the body triggered neuronal stress. We have previously demonstrated that nutrient insufficiency in neonates elicits metabolic stress in the gut. Furthermore, colostrum oxytocin modulates cellular stress response, inflammation, and autophagy markers in newborn rat gut villi prior to and after first feed. Signaling protein markers associated with the endoplasmic reticulum stress [ER chaperone binding immunoglobulin protein (BiP), eukaryotic translation initiation factor 2A (eIF2a), and eIF2a kinase protein kinase R (p-PKR)], as well as two inflammation-signaling proteins [nuclear factor-&#954;B (NF-kB) and inhibitor &#954;B (IkB)], were measured in newborn brain nuclei [nucleus of the solitary tract (NTS), paraventricular nucleus (PVN), supra-optic nucleus (SON), cortex (CX), striatum nuclei (STR), and medial preoptic nucleus (MPO)] before the first feed (unprimed by colostrum) and after the start of nursing (primed by colostrum). Expression of BiP/GRP78 and p-eIF2a were upregulated in unprimed and downregulated in primed NTS tissue. NF-kB was retained (high) in the CX, STR, and MPO cytoplasm, whereas NF-kB was lower and unchanged in NTS, PVN, and SON in both conditions. The collective BiP and p-eIF2 findings are consistent with a stress response. eIf2a was phosphorylated by dsRNA dependent kinase (p-PKR) in the SON, CX, STR, and MPO. However, in the NTS (and to a lesser extent in PVN), eIf2a was phosphorylated by another kinase, general control nonderepressible-2 kinase (GCN2). The stress-modulating mechanisms previously observed in newborn gut enterocytes appear to be mirrored in some OTR-rich brain regions. The NTS and PVN may utilize a different phosphorylation mechanism (under nutrient deficiency) from other regions and be refractory to the impact of nutrient insufficiency. Collectively, this data suggests that brain responses to nutrient insufficiency stress are offset by signaling from colostrum-primed enterocytes.

Here, we present a protocol to isolate brain nuclei in the neonatal rat brain in conjunction with first colostrum feeding. This technique allows the study of nutrient insufficiency stress in the brain as modulated by enterocyte signaling.

前に、最初の初乳の授乳後細胞ストレスと新生児ラットにおける離散のオキシトシン分泌脳核の炎症を評価します。

The goal of this protocol is to isolate oxytocin-receptor rich brain nuclei in the neonatal brain before and after first colostrum feeding. The expression ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

生体外でマウス縁帯 B 細胞のせん断流動誘起移行の解析

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...