我々は彼のリアルタイムPCR装置の使用のために博士チャールズグリーンバーグに感謝します。この作品は、MUSCホリングズがんセンタースタートアップファンド、ホリングズがんセンターACS IRG、ASCOのがん財団のキャリア開発賞を征服し、NIH 1K08HL 103780-01A1、およびNIH 3P30CA138313-01S3でサポートされています。内容はもっぱら著者の責任であり、必ずしも国立衛生研究所（NIH）やその他の資金調達エージェントの公式見解を示すものではありません。

1。骨髄細胞の単離<ol><li>男性ドナーFVB / NJマウスや頸椎脱臼続いたCO 2の方法を使用して女性のFVB / NJマウスを安楽死させる。女性FVB / NJ骨髄細胞は、競争力のある細胞として使用されます。 </li><li>マウスからの大腿骨およびtibiaesをばらばらにすると5％の熱不活性化FBSを6 mlの氷冷した1640を含む60mmの組織培養皿に置き、小さなはさみや鉗子を使用しています。筋肉や他の組織を除去するためにキムワイプティッシュを使用しています。皿の中でそれぞれの骨軸の両端を切り落とした。 </li><li> 3 ccシリンジに23G針を用いて骨の端を接続し、皿に5％熱不活化FBSを含むRPMI1640で骨髄を洗い流す。同じ針を使用して繰り返し願望によって骨髄組織を分解。 15 ml遠心チューブに細胞懸濁液を移す。 </li><li> 400 xgで5分間細胞をスピンダウンし、上清を除去し、（155 mMの電話室温赤血球溶解バッファー1mlに細胞を再懸濁otassium重炭酸塩、10mMの塩化アンモニウム、EDTA = 7.4 PHの0.1 mM）および室温で5分間インキュベートし、次に5％熱不活性化FBSを含むRPMI 1640の5-10 mlを加える。 </li><li>セルストレーナーを通してセルを渡す。新しいチューブに通る流れを収集します。 400 x gで5分間スピンダウンします。上清を除去し、細胞ペレットは、任意の赤色を含むべきではありません。赤い色の不在は、赤血球を完全に除去することを示します。 5％熱不活化FBSを含むRPMI1640 10mlに細胞ペレットを再懸濁します。細胞懸濁液は完全に均一であることを確認するために静かに渦。アリコートを取り、血球計算盤で細胞数をカウント。骨髄移植と5:2の割合で女性のライバル細胞と分量に十分な細胞やミックス男性ドナー細胞に必要な細胞のどのくらいの体積を計算します。 2×10 6個で5におけるドナー細胞の最終濃度が1×10 6個 / mlと競合他社の細胞と400 XG、PBSで洗浄し、PBSに再懸濁し、5分間スピンダウン</suP&gt; / mlである。 </li></ol> 2。競争力のある骨髄移植<ol><li>骨髄移植前に11Gy 4から6時間の単回投与における137Csのガンマ線放射体でメスのレシピエントマウス（生後8〜12週齢）を照射します。 </li><li>マウスの制止で照射雌マウスを置きます。それぞれのマウスは5×10 5ドナー細胞と2×10 5ライバル骨髄細胞を受信できるように総量0.1mlに尾静脈を介して混合ドナーと競合他社の細胞を注入します。 </li></ol> 3。試料捕集カップル·週間骨髄移植後に、麻酔条件下で眼窩出血によって女性レシピエントマウスから末梢血サンプル（〜50μl）を収集しています。サンプルは、EDTA被覆管に集められます。 BMのサンプルも記載前回と同じ手順（ステップ1）で、通常は少なくとも4ヶ月移植後、実験終了時に収集することができ、通常の20から40パーセント1脛骨骨髄細胞はDNAの十分な量を作るのに十分です。血液または骨髄サンプル年齢からマッチさせた正常雌と雄マウスも標準曲線を準備するために収集されます。 4。ゲノムDNAの単離<ol><li>各血液サンプルを室温赤血球溶解バッファー（〜200μL）の4×ボリュームを追加します。よく混ぜ、室温で5分間静置します。 1mlのPBSを追加し、溶解したRBCの大部分を除去するスピンダウンします。 </li><li>血液のDNA抽出キット（QIAmp DNAの血液抽出キット）を使用してDNAを単離する。 37℃でプレ暖かい溶出バッファー（AE）℃溶出したDNAの収量を向上させます。標準曲線のために男性と女性のDNAを同様に分離されています。 </li><li> （オプション）ゲノムDNAをさらに精製または3 M酢酸ナトリウム（pH = 5.5）の存在下でエタノール沈殿法を用いて濃縮することができる。 DNAペレットを直ちに分析のために40から50μlの蒸留水に再懸濁し、または将来の使用のために-20℃で保存されています。 </li><li> DNAのconcentrを測定ナノドロップND-1000スペクトル光度計とation。 OD 260/280 1.8から2.0の間の検体は、さらなる分析のために使用されています。 </li><li>全量50μlで4ng/μlするDNAアーゼフリーH 2 Oを用いてDNAを希釈します。 </li></ol> 5。標準曲線の準備 4 ng /μlにの濃度に男性DNAと女性のDNAを希釈し、Table1を記載のDNA混合物を作る<table border="1"><tbody><tr><td> 女性のバックグラウンドでの男性のDNAの％ </td><td> 4ng/μl男性DNAの容量（μL） </td><td> 4ng/μl女性のDNAの容量（μL） </td><td> 総容量（μL） </td></tr><tr><td> 0.2 </td><td> 1 </td><td> 499 </td><td> 500 </td></tr><tr><td> 0.5 </td><td> 1 </td><td> 199 </td><td> 200 </td></tr><tr><td> 2.5 </td><td> 5 </td><td> 195 </td><td> 200 </td></tr><tr><td> 12.5 </td><td> 25 </td><td> 175 </td><td> 200 </td></tr><tr><td> 50 </td><td> 100 </td><td> 100 </td><td> 200 </td></tr><tr><td> 87.5 </td><td> 175 </td><td> 25 </td><td> 200 </td></tr><tr><td> 100 </td><td> 200 </td><td> 0 </td><td> 200 </td></tr></tbody></table>標準曲線については、 表1。サンプル調製。 8 12wks歳の時に通常のオスとメスFVB / NJマウスを屠殺した。血液や骨髄細胞を採取した。オスとメスの細胞の細胞からDNAを単離し、そして4ng/μlの濃度で再懸濁した。雄と雌のDNAがDNA標準試料混合物を生成するために種々の割合で混合した。 6。リアルタイムPCR <ol><li>プライマー及びゲノムDNAをSYBR Green Supermixを試薬を混合することにより、PCR反応プレートを設定します。反応液量（20μL）は、各プライマーの400 nMおよび各反応における血液細胞のゲノムDNA（4ng/μlx5のμL= 20 ng）を5μlを含んでいます。 DNA SAMPレや基準は三重に設置された。プライマーの配列を表2に示す。 </li></ol><table border="1"><tbody><tr><td> 遺伝子名 </td><td> フォワード </td><td> 逆転 </td></tr><tr><td> BCL2 </td><td> 5&#39;-AAGCTGTCACAGAGGGGCTA </td><td> 5&#39;-CAGGCTGGAAGGAGAAGATG </td></tr><tr><td> Zfy1 </td><td> 5 TGGAGAGCCACAAGCTAACCA </td><td> 5&#39;-CCCAGCATGAGAAAGATTCTTC </td></tr></tbody></table>ネズミBCL2とZfy1については、 表2のRT-PCRプライマー配列。 <ol start="2"><li> 95℃で3分間、融解曲線が続く10秒間、95℃、58℃25秒、72℃15秒→42増幅サイクル：以下の条件をバイオラッドiQ5 PCR装置を用いてPCR反応を行うステップ。 </li><li> Bio-RadのiQ5 2.1 Standard Editionの光学系によってサイクル閾値（Ct）値を取得します。計算ΔCT（CT Zfy1 &lt;/ sup&gt;の-CT BCL2）値、Zfy1発現レベルは2-δCtの値として計算されます。 </li><li>既知のオス/メスの標準混合物から2-ΔCT読書を使用して、それぞれの反応シリーズの標準曲線を確立します。標準曲線は、線形回帰フィッティングとの混合物で知ら％男性DNAに三重の2ΔCT値の平均値をプロットすることによって生成されます。 </li></ol>

<ol>
	<li>McCulloch, E. A., Till, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+radiation+sensitivity+of+normal+mouse+bone+marrow+cells,+determined+by+quantitative+marrow+transplantation+into+irradiated+mice.">The radiation sensitivity of normal mouse bone marrow cells, determined by quantitative marrow transplantation into irradiated mice.</a> Radiat. Res. 13, 115-125 (1960).</li><li>Xiao, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cells+lacking+Ott1+display+aspects+associated+with+aging+and+are+unable+to+maintain+quiescence+during+proliferative+stress.">Hematopoietic stem cells lacking Ott1 display aspects associated with aging and are unable to maintain quiescence during proliferative stress.</a> Blood. 119, 4898-4907 (2012).</li><li>Kang, Y., Chen, B. J., Deoliveira, D., Mito, J., Chao, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+enhancement+of+donor+hematopoietic+cell+engraftment+by+the+CXCR4+antagonist+AMD3100+in+a+mouse+transplantation+model.">Selective enhancement of donor hematopoietic cell engraftment by the CXCR4 antagonist AMD3100 in a mouse transplantation model.</a> PLoS One. 5, e11316 (2010).</li><li>Sadeghi, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GVHD+after+chemotherapy+conditioning+in+allogeneic+transplanted+mice.">GVHD after chemotherapy conditioning in allogeneic transplanted mice.</a> Bone Marrow Transplant. 42, 807-818 (2008).</li><li>Taketo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FVB/N:+an+inbred+mouse+strain+preferable+for+transgenic+analyses.">FVB/N: an inbred mouse strain preferable for transgenic analyses.</a> Proc. Natl. Acad. Sci. U.S.A. 88, 2065-2069 (1991).</li><li>Morisaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotypic+analysis+using+a+Y-chromosome-specific+probe+following+bone+marrow+transplantation.">Genotypic analysis using a Y-chromosome-specific probe following bone marrow transplantation.</a> Am. J. Hematol. 27, 30-33 (1988).</li><li>Lo, Y. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+fetal+DNA+in+maternal+plasma+and+serum:+implications+for+noninvasive+prenatal+diagnosis.">Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis.</a> Am. J. Hum. Genet. 62, 768-775 (1998).</li><li>Byrne, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chimerism+analysis+in+sex-mismatched+murine+transplantation+using+quantitative+real-time+PCR.">Chimerism analysis in sex-mismatched murine transplantation using quantitative real-time PCR.</a> Biotechniques. 32, 279-280 (2002).</li><li>Ma, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+recipient-derived+cells+in+allograft+neointima+formation+and+the+response+to+stent+implantation.">Contribution of recipient-derived cells in allograft neointima formation and the response to stent implantation.</a> PLoS One. 3, e1894 (2008).</li><li>Bosio, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+between+real-time+quantitative+PCR+and+DNA+hybridization+for+quantitation+of+male+DNA+following+myoblast+transplantation.">A comparison between real-time quantitative PCR and DNA hybridization for quantitation of male DNA following myoblast transplantation.</a> Cell Transplant. 13, 817-821 (2004).</li><li>Merchant, A., Joseph, G., Wang, Q., Brennan, S., Matsui, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gli1+regulates+the+proliferation+and+differentiation+of+HSCs+and+myeloid+progenitors.">Gli1 regulates the proliferation and differentiation of HSCs and myeloid progenitors.</a> Blood. 115, 2391-2396 (2010).</li><li>Nagamine, C. M., Chan, K., Hake, L. E., Lau, Y. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+two+candidate+testis-determining+Y+genes+(Zfy-1+and+Zfy-2)+are+differentially+expressed+in+fetal+and+adult+mouse+tissues.">The two candidate testis-determining Y genes (Zfy-1 and Zfy-2) are differentially expressed in fetal and adult mouse tissues.</a> Genes &amp; development. 4, 63-74 (1990).</li><li>Grundler, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+PIM+serine/threonine+kinases+in+FLT3-ITD-induced+leukemogenesis+reveals+PIM1+as+regulator+of+CXCL12-CXCR4-mediated+homing+and+migration.">Dissection of PIM serine/threonine kinases in FLT3-ITD-induced leukemogenesis reveals PIM1 as regulator of CXCL12-CXCR4-mediated homing and migration.</a> J. Exp. Med. 206, 1957-1970 (2009).</li></ol>

我々は、開示することなく競合する金融利害関係はありません。

我々の現在の研究の目的は、観客に競争力のあるマウス骨髄移植モデルのドナー細胞の生着を​​定量化するためのPCRベースの技術を提供することにある。いくつかの研究は移植モデル9-10にドナー細胞を検出するためにRT-PCRを用いて報告されている。博士シュワルツェンバーガーのグループは、最初のY染色体に特異的な8を増幅するリアルタイムPCRを用いてマウス骨髄移植モ...

マウス骨髄（BM）移植モデルは、1960年代に最初1で開発されました。このモデルは広範囲にホストレシピエントマウスにおけるドナーの造血幹細胞（HSC）の生物学の研究に用いられてきた。マウス骨髄移植モデルは、HSCの機能とその調節に関する貴重な知識を提供してくれましたし、HSCの研究に不可欠である。 C56Bl/6J CD45.2-B6.SJL CD45.1様C57BL/6J H2B-のBalb / C H2Dまたはジェニック移植モデルのような同種骨髄移植モデルでは、効果の、HSCの活性が2日に遺伝子機能を研究するために多くの研究室で使用されているそのような移植片対宿主病（GVHD）4などのHSC機能3または移植関連疾患に対する薬物治療。 そのようなMHCハプロタイプまたはCD45.1として細胞表面マーカーは、一般的に受信者から発信された細胞から区別するドナー由来細胞に使用されます。 C57BL/6J H2B、CD45.2 </suドナー細胞の寄与を簡単CD45.1 対 CD45.2またはH2B対を測定するフローサイトメトリーにより評価することができるので、p&gt;は、BALB / C H2DとB6.SJL CD45.1は、骨髄移植で最も一般的に使用されるマウス株であるH2D。しかし、このようなFVB / NJ 5とC3Hのような他の多くの菌株もしばしば遺伝子組み換えトランスジェニックやノックアウトマウスを生成するために使用されます。これらのマウスは近交系に戻し交配および混合MHC /遺伝的背景で維持することができる。ドナー特異的細胞表面マーカーが使用できない場合があり、これらの例では、ドナー細胞の生着とHSC機能を決定することは難しいかもしれません。 性別不適合骨髄移植におけるサザンブロットによってドナー雄性細胞を検出するために、Y染色体特異的DNAプローブを用いて、前記第三輪博士のグループ6によって開発されました。その後、性決定領域YのためのリアルタイムPCRはmaを定量するため、正確かつ特異性の高い方法であることが判明した母体血システム7のLe胎児細胞。この概念は、ドナー細胞の生着8を決定するために、マウス骨髄移植モデルにおけるリアルタイムPCR法の開発のための博士シュワルツェンバーガーのグループが適応されました。我々はさらにFVB / NJマウス骨髄移植モデルのドナー細胞の生着を​​測定するためのこの方法を変更しました。このメソッドは、現在広くHSCの生物学におけるPIM1キナーゼの役割を研究するために我々のグループで利用されている。

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td> RPMI 1640 </td><td> Hyclone社</td><td> SH30255.01 </td><td></td></tr><tr><td> FBS </td><td>インビトロジェン</td><td> 16140-017 </td><td>不活性化された熱</td></tr><tr><td>塩化アンモニウム</td><td> MP Biomedicaals </td><td> 194806 </td><td></td></tr><tr><td>炭酸水素カリウム</td><td>フィッシャー</td><td> P184-500 </td><td></td></tr><tr><td> QIAamp調製DNAは血液minikit </td><td>キアゲン</td><td> 51106 </td><td></td></tr><tr><td>セルストレーナー</td><td>バイオサイエンスのBD </td><td></td><td></td></tr><tr><td> IQ SYBR Green Supermixを</td><td>バイオラッド</td><td> 170-8882 </td><td></td></tr><tr><td> iQ5リアルタイムPCR装置</td><td>バイオラッド</td><td></td><td></td></tr><tr><td>スペクトル光度計</td><td>ナノドロップND-1000 </td><td> ND-1000 </td><td></td></tr></tbody></table>

using quantitative real-time pcr to determine donor cell engraftment in a competitive murine bone marrow transplantation model

マウス骨髄移植モデルは、造血幹細胞（HSC）の機能および決定遺伝子/ HSCを調節する分子を測定する際の重要なツールを提供します。これらの移植モデルシステムでは、造血幹細胞の機能が生着し、再構成する致死量の放射線を照射したレシピエントマウスにこれらの細胞の能力によって決定されます。一般的には、ドナー細胞の寄与/生着は、フローサイトメトリーを用いたドナー特異的な細胞表面タンパク質に対する抗体によって測定されます。しかし、この方法は、大きく特異性とすべてのマウス系統では使用できない場合があり、受信者から発信された細胞からドナー由来細胞を区別するために、細胞表面マーカーの能力に依存します。市場では遺伝的に改変されたマウス系統の様々な背景を考えると、この細胞表面/フローサイトメトリーベースの方法は、特にジェニック受け手CEから独立したドナー細胞に明確に定義された表面マーカーを欠いているマウス系統に重大な制限がありますLLS。ここでは、移植レシピエントマウスにおけるドナー細胞の生着/寄与を決定するために、PCRベースの手法を報告した。私たちは、致死量の放射線を照射ジェニック雌マウスに男性ドナー骨髄の造血幹細胞を移植した。末梢血試料を異なる時点の移植後に採取した。骨髄サンプルは、実験の終了時に得られた。ゲノムDNAを単離し、Y染色体特異的遺伝子、Zfy1は、定量的リアルタイムPCRを用いて増幅した。女性レシピエントマウスにおける男性ドナー由来細胞の生着は、男性対女性のDNAの既知の割合で標準曲線に対して算出した。 BCL2は、総DNA量を正規化するために参照遺伝子として用いた。我々のデータは、このアプローチは確実にドナー細胞の生着を​​決定し、マウス骨髄移植モデルにおける造血細胞再構成を測定するのに有用な、まだ簡単な方法を提供することが示唆された。本手法は、日常的にほとんどの研究室で行うことができるため、ノーこのようなフローサイトメトリーなどの高価な機器が必要です。

図1及び図2は、標準曲線の例は男性DNAの割合に対して2-ΔCTの平均値でプロットを示した。 図1Aは 、それぞれ78.5℃、88.5℃でローカライズBCL2とZfy1リコンのための特異的な融解温度を示した。 BCL2は、各PCR反応にロードされたDNAの総量を正規化するために参照遺伝子として使用されます。各標準サンプルのためBCL2増幅曲線は、（ログビュー）ロードされたDNA（ <...

Watch this Scientific Journal Video about Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model at JoVE.com

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

ドナー細胞の生着を​​決定することは、明確に定義された表現型マーカーを欠くマウス骨髄移植モデルにおける課題を提示する。私たちは、女性の移植レシピエントマウスに男性ドナー細胞の生着を​​定量化するための方法論を説明した。このメソッドは、HSCの機能の研究のためにすべてのマウス系統で使用することができます。

競争力のあるマウス骨髄移植モデルにおけるドナー細胞の生着を​​決定するために、定量的リアルタイムPCRを用いて

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

using-quantitative-real-time-pcr-to-determine-donor-cell-engraftment

Research

JoVE Journal

Medicine

15.3K ビュー.  Medical University of South Carolina. ドナー細胞の生着を​​決定することは、明確に定義された表現型マーカーを欠くマウス骨髄移植モデルにおける課題を提示する。私たちは、女性の移植レシピエントマウスに男性ドナー細胞の生着を​​定量化するための方法論を説明した。このメソッドは、HSCの機能の研究のためにすべてのマウス系統で使用することができます。

ビデオ: Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

のマウスモデル子宮内で移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

医学

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

定量的リアルタイムPCR（定量PCR）による前立腺腫瘍におけるマイクロRNAの検出

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

急性白血病の治療のためのチロシンキナーゼ阻害剤の前臨床評価

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

変更されたカフ法を用いたマウス子宮頸心臓移植モデル

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

初代ヒト骨髄およびその悪性腫瘍を研究する三次元組織培養モデル

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

定量リアルタイムPCRを用いて脳脊髄液マイクロRNAプロファイリング

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

ネズミ角膜移植：固体臓器移植の最も一般的な形態を研究するためのモデル

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

定量的リアルタイムPCRによるマウス乳腺腫瘍モデルにおける肺転移の評価

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation.
Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.

改変非縫合カフ技術を用いたマウス子宮頸動脈大動脈移植モデル

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.

競争力のあるマウス骨髄移植モデルにおけるドナー細胞の生着を​​決定するために、定量的リアルタイムPCRを用いて

競争力のあるマウス骨髄移植モデルにおけるドナー細胞の生着を決定するために、定量的リアルタイムPCRを用いて