Name: determination of sialic acids in liver and milk samples of wild-type and cmah knock-out mice.
Uploaded: 2017-07-14T15:00:00.000+00:00
Duration: 8 min 4 s
Description: Watch this Scientific Journal Video about Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice. at JoVE.com

この研究は、中国の自然科学財団（助成金番号31471703、A0201300537および31671854、JVおよびLL）および100の外国人才能計画（助成番号JSB2014012からJV）によって部分的に支持された。

動物被験者に関する手順は、SPFに収容された動物を用いて、実験的動物福祉に関する国家ガイドライン（科学技術省、中国、2006年）に従って、南京農業大学の実験動物センターの倫理委員会によって承認されている施設（許可ID：SYXK-J-2011-0037）。 1. Cmahノックアウトマウスモデル<ol><li>揚州大学（中国）の比較医学センターの野生型C57Bl / 6マウスを使用する。 注：CMAHノックアウトマウスもあるCMAH遺伝子のエクソン6から92個の塩基対を除去することによって商業的にCRISPR / Cas9 20ストラテジーを使用して前CMAHノックアウト研究17、19及び得られたのから提供された情報に基づいて生成されましたヒトCMAH遺伝子で欠失している19 。正のF 0 &lt;/サブ&gt; -mice（2人）は、ヘテロ接合F 1 -miceを得るために、野生型マウスと交配しました。ヘテロ接合F 1 -mice（5個体）と交差した後、ホモ接合CMAHノックアウトF 2 -mice正常（3個体）を得ることができました。 </li><li> 市販のDNA精製キットを用いて、Zangala 21で示される方法に従ってゲノムDNAを用いてマウスの遺伝子型決定を行う。 <ol><li>市販のDNAポリメラーゼおよびプライマー対5&#39;gaaagggctcggctctgtatgaa3 &#39;および5&#39;tttaaagtcccgggtgagaagc3&#39;を用いて、 Cmah遺伝子の対応するDNA断片を増幅する。 94℃で40秒間の変性、63℃で40秒間のアニーリング、72℃で1分間の伸長からなる34回のPCRサイクルを用いて遺伝子増幅を行う。 </li><li> PCR産物を1％アガロースゲルで視覚化する。野生型個体については単一のバンド（0.5kb）を、野生型個体については二重バンド（0.4および0.5kb）を観察すべきである。ヘテロ接合性ノックアウト個体、およびホモ接合性ノックアウト個体の単一バンド（0.4kb）を含む。 </li></ol></li></ol> 2.サンプル収集<ol><li> Willingham らによって記載されたプロトコルを用いてマウスミルクを収集する。 22 。 </li><li>ゴンザレス（Gonçalves） らによって記載されたプロトコールを用いてマウス肝臓組織を収集する。 -80℃で23や店舗サンプル。 </li></ol>ミルクからのシアル酸の単離<ol><li>蒸留H 2 Oの88ミリリットルに氷酢酸12ミリリットルを添加することにより、2 M酢酸溶液100mLを調製</li><li>ミルク50μLを1.5 mL遠心チューブに移し、調製した酢酸水溶液1.2 mLを加えます。 </li><li>懸濁液を80℃で4時間インキュベートし、14,000 xgで10分間遠心する。 </li><li>上清のトップ1,000μLを新鮮な1.5mL遠心分離管に移し、rem室温で遠心蒸発によって溶媒を除去して乾燥させる（付着した真空ポンプに応じて、これは4〜20時間かかる）。蒸留H 2 Oの500μlのサンプルを再溶解</li><li> マイクロ陰イオン交換カラムを準備する。この工程は、アニオン性化合物を特異的に富化し、カゼインおよび中性化合物をミルクおよび肝臓サンプルから除去し、したがってシアル酸誘導体化のための蛍光発生OPD標識剤の選択性を増加させる。 <ol><li>陰イオン交換樹脂（Dowex 1X8）200 mgを空の3 mLカラムチューブに移して、それぞれのサンプルを移します。 </li><li>ステップ3.1で調製した2mLの酢酸水溶液を加えて、樹脂中の塩化物イオンを酢酸イオンで置換する。溶剤が樹脂の上部に達するとすぐに、コックを閉じます。 </li><li>優しく、蒸留H 2 Oの2 mLを加えストップコックを開いて、すぐに溶媒に達するの最もとして流れを止めます樹脂の頂部。樹脂が常に溶媒で覆われていることを確認してください。 </li><li>過剰の酢酸を除去するための蒸留H 2 O 2mLで樹脂カラムを2回洗浄します。 </li></ol></li><li>ステップ3.4のサンプル溶媒を500μL移します。樹脂カラム上に流し、フロースルーを廃棄する。穏やか1.5mLの中に50mMの酢酸アンモニウム溶液（蒸留H 2 O 100mLに溶解し、酢酸アンモニウムの386 mg）を1mLのを用いて蒸留H 2 Oで溶出2mLで樹脂からサンプルアニオンを樹脂を洗浄遠心チューブ。室温で遠心蒸発によって溶媒を除去して乾燥させる。 注：乾燥サンプルは、4〜6℃で12ヶ月まで保存できます。 </li></ol>肝臓組織からのシアル酸の単離<ol><li>マウス肝臓20〜50 mgを静かに解凍し、Dounce組織粉砕機（1 mLまたは2 mLの容量）に移します。 1.2mLの酢酸水溶液ステップ3.1で調製し、10秒間穏やかに剪断することによって組織をホモジナイズする。得られた懸濁液を1.5mLの遠心管に移す。 </li><li>ステップ3.3に従ってください。 3.6まで。 注：乾燥サンプルは、4〜6℃で12ヶ月まで保存できます。 </li></ol> 5.混合シアル酸標準液の調製<ol><li>分析用天秤に5〜8mgのN-アセチルノイラミン酸を1.5mL遠心分離管に量り入れる。正確な重量に注意し、各mgについて164μLの蒸留H 2 Oを加える（ すなわち 、Neu5Acの重量が7.2mgである場合、7.2x164 =1,181μLの蒸留H 2 Oを加える）。この結果、20mM Neu5Acストック溶液が得られ、-20℃で最大12ヶ月保存することができます。 </li><li> 1mgアリコートなどの中程度の価格で、より少量のN-グリコリルノイラミン酸を得る。約20mMのNeu5Gcストック溶液を得るために、154μLの蒸留H 2 Oを化合物に直接添加する。転送する1.5mLの遠心チューブに加えた。この溶液は、-20℃で最大12ヶ月間保存することができます。 </li><li>ステップ5.1および5.2の各ストック溶液から5μLを新鮮な1.5mL遠心分離チューブに混合し、室温で乾燥させる遠心蒸発によって溶媒を除去する。 注：混合シアル酸標準は、4〜6℃で最大12ヶ月間保存することができます。 </li></ol>シアル酸の蛍光誘導体化<ol><li> 100mLのo-フェニレンジアミンおよび208mgの亜硫酸水素ナトリウムからなる10mLのOPD溶液を10mLの蒸留H 2 O中に調製する。 </li><li>マウスミルク（ステップ3）、マウス肝臓（ステップ4）、または混合シアル酸標準（ステップ5）に由来するシアル酸サンプルに20μLのOPD溶液を加える。 30秒間激しくボルテックスし、サンプルを暗所で80℃で4時間インキュベートする（ すなわち 、マイクロチューブをアルミニウム箔で包む）。 </li><li>サンプルを5分間冷却し、80μ、蒸留H 2 OのL 1分間14,000×gでチューブを遠心分離。 </li><li>上清から80μLを300μL高回収率HPLCバイアルに移す。誘導体化されたシアル酸サンプルは、4〜6℃で最大1週間保存することができます。 </li></ol>シアル酸誘導体のHPLC分析<ol><li>オンライン蛍光検出器に接続された標準HPLCシステムを使用してサンプルを分析する。 </li><li>分析には、長さ250mm、直径4.6mmの標準寸法の逆相C18カラムを使用します。 </li><li> 200mLのストック溶液を800mLのLCMSグレード水で希釈することにより溶媒Aを調製する（LCMS-液体クロマトグラフィー質量分析法）。原液自体は以下のように調製することができる： <ol><li> 46gのギ酸を800mLのLCMSグレードのH 2 Oに加える。 </li><li>水酸化アンモニウム溶液（puriss.pa）を滴下してpHを4.5に調整する。 </li><li>溶媒をLCMSグレードのH 2 Oで1,000mLまで充填してください。このストック溶液は、4〜6℃で3ヶ月まで保存できます。 </li></ol></li><li>溶媒Bについては、LCMS級アセトニトリルを使用します。 </li><li> 次の勾配溶離を用いて1mL /分の流速でシアル酸誘導体を分離する： <ol><li>まず溶剤Aに混合した溶剤Bを10％加える。 </li><li> 0分から15分まで、直線勾配で60％まで溶媒Bの割合を徐々に増加させる。 </li><li> 15分から16分まで、溶媒Bの割合を60％から90％の直線勾配で急速に増加させる。これにより、HPLCカラムの洗浄​​が開始される。 </li><li> HPLCカラムをさらに洗浄するには、溶媒Bのレベルを16分〜18分の間に90％に保ち、その後溶媒Bのレベルを18分〜19分の間に徐々に10％に徐々に低下させる。 </li><li> HPLCカラムを19〜24分の10％Bの開始条件に再平衡させる。 </li></ol></li><li>に50μLのサンプルをHPLCシステムに注入する。 </li><li> 373 / 488nmの蛍光検出器励起/発光波長を用いて溶離液をモニターし、誘導体化されたシアル酸は、約9分（Neu5Gc-OPDについて）および10分（Neu5Ac-OPDについて）の予想保持時間で期待することができる。 </li><li> F Neu5Gc [％] = 100×A Neu5Gc /（A Neu5Ac + A Neu5Gc ）のように、Neu5Ac（A Neu5Ac ）およびNeu5Gc（A Neu5Gc ）の蛍光ピーク面積からのNeu5Gc（F Neu5Gc ）の相対量を計算する。ヘテロ接合性及び野生型マウスのF のNeu5Gcの値はマウスの年齢と2％の間の組織のタイプ（に応じて広範囲に変化し得るのに対し、ホモ接合CMAHノックアウトマウスからミルクおよび肝臓のサンプルの場合にはF のNeu5Gcは 、0％であるべきですおよび&gt; 90％）、予測誤差マージンは±12％です。 </li></ol>

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本明細書に提示されたプロトコールは、乳及び肝臓試料のNeu5Gcの相対量を分析及び定量することにより、ホモ接合Cmahノックアウトマウスの表現型評価を可能にする。分析は、蛍光検出を伴う標準的なHPLC設定を用いて行った。この手順の最も重要なステップは、陰イオン交換カラムの調製および陰イオン交換クロマトグラフィーの実施である。樹脂を適切に沈殿させ、適切な洗浄および溶出画分を集めることは、少しの練習を要する。 あるいは、本明細書で使用する誘導体化剤OPDは、より高価な誘導体化剤DMB（1,2-ジアミノ-4,5-メチレンジオキシベンゼン）で容易に置換することができる。さらに、得られたシアル酸誘導体を工程6.5から分析した。蛍光検出（ すなわち 、MS-2020検出器と結合したShimadzu Nexera UPLCシステム）の代わりに質量分析検出を用いて分析することもできる。 Aステップ7.2-7.6を適用すると、382.0および398.0のm / z比（これらはそれぞれ、Neu5Ac-OPDおよびNeu5Gc-OPDのH + -付加物である）の正イオン化モード走査を用いてシアル酸誘導体を直接検出する。 この方法の1つの限界は、Neu5Gc含量がNeu5Ac量に対してのみ定量化されるが、絶対的には定量化されないことである。そのためには、試料調製の初期段階で、内部標準として明確で定量可能なシアル酸を添加する必要があります。別の欠点は、Neu5Gcヘテロ接合ノックアウトマウスの相対的な量が、個々のマウスの試料の種類（ すなわち組織）および年齢に依存して強く変化し得ることから、ホモ接合であるがヘテロ接合のCmahノックアウトマウスのみが明白に同定され得ることである。この方法の今後の発展には、内部標準の添加およびマウスにおけるシアル酸の絶対定量化が含まれ得る。

N-アセチルノイラミン酸（Neu5Ac）およびN-グリコリルノイラミン酸（Neu5Gc）は、ほとんどの哺乳動物において最も一般的なシアル酸である1 。内因性のNeu5Acを合成することができるが、人間が原因CMP-Neu5Acをヒドロキシラーゼ2、3 CMAHをコードする遺伝子上の主要エクソン欠失へのNeu5Gcを産生することができません。しかし、動物性食品は、抗のNeu5Gc抗体の産生につながるのNeu5Gc 4、5、6の栄養源となり、したがってのNeu5Gc 7に対する免疫応答を誘発することができます。 Neu5Gcこの食物効果は、慢性炎症および種々の他の疾患8、9、10に寄与することが疑われます。 hでのNeu5Gcの効果を包括的に理解するために食品由来のシアル酸の影響を体系的に研究するための動物モデルが非常に望ましい。ノックアウトマウスの解析のためのポリメラーゼ連鎖反応（PCR）に基づくプロトコールは十分に確立されており、遺伝子型評価のための簡便な方法であるが、代謝レベルでの表現型の機能解析にはより特異的な分析方法が必要である。 Cmahノックアウトマウスモデルの表現型は、肝臓または乳試料中のシアル酸の組成物を単離および分析することによって評価することができる。シアル酸をレゾルシノール11またはチオバルビツール酸12と反応させると、発色団生成物が形成され、プレートリーダベースの設定を用いて簡単に分析することができるが、シアル酸のみを分析することができる酸含量を決定することができる。あるいは、シアル酸の分析は、ガスクロマトグラフィー13 、MALDI-ToF質量分析14または電流測定法15 。しかし、最も一般的に適用されるシアル酸分析方法は、加水分解放出に基づく蛍光誘導体化およびその後の高速液体クロマトグラフィー16、17、18が続きます。

<table><tbody><tr><td>&lt;strong&gt;Chemicals:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>N-acetylneuraminic acid</td><td>Sigma</td><td>A0812</td><td></td></tr><tr><td>N-glycolylneuraminic acid</td><td>Sigma</td><td>50644</td><td>1 mg aliquot should be sufficient</td></tr><tr><td>o-Phenylenediamine</td><td>Sigma</td><td>694975</td><td></td></tr><tr><td>Sodium hydrogen sulfite</td><td>J&amp;amp;K Scientific Ltd</td><td>75234</td><td></td></tr><tr><td>&lt;strong&gt;Tools/Materials:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>3 mL SPE tubes</td><td>Supelco</td><td>Sigma 57024</td><td>empty solid phase extraction columns</td></tr><tr><td>Luer stopcock</td><td>Sigma</td><td>S7396</td><td>to stop the flow of the SPE tube</td></tr><tr><td>Dowex 1X8</td><td>Dow Chemicals</td><td>Sigma 44340</td><td>200 - 400 mesh</td></tr><tr><td>Dounce tissue grinder</td><td>Sigma</td><td>D8938</td><td>tight fit</td></tr><tr><td>&lt;strong&gt;HPLC Analysis:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>High-recovery HPLC vial</td><td>Agilent Technologies</td><td>#5188-2788</td><td></td></tr><tr><td>HPLC System</td><td>Shimadzu</td><td>Nexera</td><td></td></tr><tr><td>Fluorescence Detector for HPLC</td><td>Shimadzu</td><td>RF-20Axs</td><td></td></tr><tr><td>HPLC Column</td><td>Phenomenex</td><td>Hyperclone ODS</td><td>250 x 4.6 mm</td></tr><tr><td>LCMS-grade H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck Millipore</td><td>#WX00011</td><td></td></tr><tr><td>LCMS-grade Acetonitrile</td><td>Merck Millipore</td><td>#100029</td><td>Hypergrade</td></tr><tr><td>Ammonium hydroxide solution</td><td>Fluka</td><td>#44273</td><td>puriss. P.a.</td></tr></tbody></table>

determination of sialic acids in liver and milk samples of wild-type and cmah knock-out mice.

CMAH（シチジンモノホスフェート-N-アセチルノイラミン酸ヒドロキシラーゼ）は、哺乳動物におけるシチジンモノホスフェート-N-アセチルノイラミン酸の酸化を担う。しかしながら、ヒトは、 CMAH遺伝子の一次エクソン欠失のために、シチジンモノホスフェート-N-アセチルノイラミン酸をシチジンモノホスフェート-N-グリコリルノイラミン酸に酸化することができない。 CMAH活性の欠如の影響および影響をより詳細に理解するために、マウスにおけるCmahノックアウトモデルは、基礎および応用研究に非常に興味深い。このマウスモデルの表現型を決定するための分析方法は、本明細書中に詳細に記載されており、野生型およびCmahノックアウトマウスの肝臓および乳汁中のN-アセチルノイラミン酸およびN-グリコリルウラミン酸の両方の検出に基づく。内因性シアル酸は、放出され、o-フェニレンジアミンで誘導体化されて蛍光発生誘導体を生成し、その後、HPLCによって分析することができる。提示されたプロトコルは、様々な他の起源の乳および組織サンプルの分析にも適用され、N-グリコリルノイラミン酸の栄養および健康への影響を調査するのに有用である可能性がある。

記載された分析方法の概略図は図1に示されており、野生型およびCmahノックアウト突然変異体マウスの乳および肝臓サンプルからのシアル酸の単離およびこれらの成分の蛍光誘導体化およびHPLC分析が含まれる。 図2および図3は、ホモおよびヘテロ接合ノックアウトCmahマウス（ - / - および+/-）および野生型マウス由来の乳および肝臓試料の誘導体化シアル酸の代表的なHPLCクロマトグラムを示す。 図4は、HPLCピーク面積から計算した分析マウス試料のN-グリコリルノイラミン酸（Neu5Gc）の相対量を示す。 <img alt="図1" class="xfigimg" src="/files/ftp_upload/56030/56030fig1.jpg" /> 図 <stro<html> ng class = &quot;xfig&quot;&gt; 1 ：マウス由来の乳および肝臓試料からのシアル酸について記載された分析方法の概略図。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56030/56030fig1large.jpg" target="_blank">この図の拡大版を見るには、ここをクリックしてください。</a> <img alt="図2" class="xfigimg" src="/files/ftp_upload/56030/56030fig2.jpg" /> 図2 ： ホモおよびヘテロ接合性ノックアウトCmahマウス（ - / - および+/-）および野生型マウス由来の乳サンプルからの蛍光標識シアル酸のHPLCクロマトグラム。上部のクロマトグラムは混合シアル酸標準です。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56030/56030fig2large.jpg" target="_blank">この図の拡大版を見るには、ここをクリックしてください。</a> &quot;src =&quot; / files / ftp_upload / 56030 / 56030fig3.jpg &quot;/&gt; 図3 ： ホモおよびヘテロ接合性ノックアウトCmahマウス（ - / - および+/-）および野生型マウス由来の肝臓試料からの蛍光標識シアル酸のHPLCクロマトグラム。上部のクロマトグラムは混合シアル酸標準です。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56030/56030fig3large.jpg" target="_blank">この図の拡大版を見るには、ここをクリックしてください。</a> <img alt="図4" class="xfigimg" src="/files/ftp_upload/56030/56030fig4.jpg" /> 図4 ： HPLCピーク領域から計算した分析マウスサンプルのN-グリコリルノイラミン酸（Neu5Gc）の相対量。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56030/56030fig4large.jpg" target="_blank">この図の拡大版を見るには、ここをクリックしてください。</a>

Watch this Scientific Journal Video about Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice. at JoVE.com

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

我々は、マウス肝臓および牛乳中のN-アセチルノイラミン酸およびN-グリコリルウラミン酸の定量のためのHPLCベースの方法を記載する。

野生型および肝臓の肝臓試料および牛乳試料中のシアル酸の定量<em&gt; CMAH</em&gt;ノックアウトマウス。

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

determination-sialic-acids-liver-milk-samples-wild-type-cmah-knock

Nanyang Technological University

Research

JoVE Journal

Biology

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

9.7K ビュー.  Nanjing Agricultural University. 我々は、マウス肝臓および牛乳中のN-アセチルノイラミン酸およびN-グリコリルウラミン酸の定量のためのHPLCベースの方法を記載する。 

ビデオ: Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

マウス肝臓における胆管密度の決定

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

発生生物学

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing termini of oligo- and polysaccharides, as well as its unique chemical characteristics, sialic acid is involved in a multitude of different receptor-ligand interactions. By modifying the expression of sialic acid on the cell surface, sialic acid-dependent interactions will consequently be influenced. This can be helpful to investigate sialic acid-dependent interactions and has the potential to influence certain diseases in a beneficial way. Via metabolic glycoengineering (MGE), the expression of sialic acid on the cell surface can be modulated. Herein, cells, tissues, or even entire animals are treated with C2-modified derivatives of N-acetylmannosamine (ManNAc). These amino sugars act as sialic acid precursor molecules and therefore are metabolized to the corresponding sialic acid species and expressed on glycoconjugates. Applying this method produces intriguing effects on various biological processes. For example, it can drastically reduce&#160;the expression of polysialic acid (polySia) in treated neuronal cells and thus affects neuronal growth and differentiation. Here, we show the chemical synthesis of two of the most common C2-modified N-acylmannosamine derivatives, N-propionylmannosamine (ManNProp) as well as N-butanoylmannosamine (ManNBut), and further show how these non-natural amino sugars can be applied in cell culture experiments. The expression of modified sialic acid species is quantified by high performance liquid chromatography (HPLC) and further analyzed via mass spectrometry. The effects on polysialic acid expression are elucidated via Western blot using a commercially available polysialic acid antibody.

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid is a typical monosaccharide-unit found in glycoconjugates. It is involved in a plethora of molecular and cellular interactions. Here we present a method to modify cell surface sialic acid expression using metabolic glycoengineering with N-acetylmannosamine derivatives.

Nを用いたシアル酸の代謝独自-アシル-Mannosamines を変更

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing ...

生物工学

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

C57BL / 6マウスにおける全身性エリテマトーデス（SLE）のBM12誘導モデル

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible ...

医学

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

マウスの唾液腺機能の測定法

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

免疫・感染学

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' pathogens (Listeria monocytogenes, Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, and Rickettsia spp.) gain access to the infected cell cytosol by physically and enzymatically degrading the primary vacuolar membrane. Once in the cytosol, these pathogens both proliferate as well as generate sufficient mechanical forces to penetrate the plasma membrane of the host cell in order to infect new cells. Here, we show how this terminal step of the cellular infection cycle of L. monocytogenes (Lm) can be quantified by both colony-forming unit assays and flow cytometry and give examples of how both pathogen- and host-encoded factors impact this process. We also show a close correspondence of Lm infection dynamics of cultured cells infected in vitro and those of hepatic cells derived from mice infected in vivo. These function-based assays are relatively simple and can be readily scaled up for discovery-based high-throughput screens for modulators of eukaryotic cell function.

We describe both in vitro and in vivo infection assays that can be used to analyze the activities of host-encoding factors.

プローブに細菌の病原体を用いた細胞・生体レベルのホスト応答

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution

Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight1-3. Secreted mucins, produced by goblet cells and the gastric mucosa, provide the scaffold for a micrometers-thick mucus layer that lines the epithelia of the gut and respiratory tract3,4. In addition to mucins, mucus layers also contain antimicrobial peptides, cytokines, and immunoglobulins5-9. The mucus layer is an important part of host innate immunity, and forms the first line of defense against invading microorganisms8,10-12. As such, the mucus is subject to numerous interactions with microbes, both pathogens and symbionts, and secreted mucins form an important interface for these interactions. The study of such biological interactions usually involves histological methods for tissue collection and staining. The two most commonly used histological methods for tissue collection and preservation in the clinic and in research laboratories are: formalin fixation followed by paraffin embedding, and tissue freezing, followed by embedding in cryo-protectant media.
Paraffin-embedded tissue samples produce sections with optimal qualities for histological visualization including clarity and well-defined morphology. However, during the paraffin embedding process a number of epitopes become altered and in order to study these epitopes, tissue sections have to be further processed with one of many epitope retrieval methods13. Secreted mucins and lipids are extracted from the tissue during the paraffin-embedding clearing step, which requires prolong incubation with organic solvents (xylene or Citrisolv). Therefore this approach is sub-optimal for studies focusing on the nature and distribution of mucins and mucus in vivo.
In contrast, freezing tissues in Optimal Cutting Temperature (OCT) embedding medium avoids dehydration and clearing of the sample, and maintains the sample hydration. This allows for better preservation of the hydrated mucus layer, and thus permits the study of the numerous roles of mucins in epithelial biology. As this method requires minimal processing of the tissue, the tissue is preserved in a more natural state. Therefore frozen tissues sections do not require any additional processing prior to staining and can be readily analyzed using immunohistochemistry methods.
We demonstrate the preservation of micrometers-thick secreted mucus layer in frozen colon samples. This layer is drastically reduced when the same tissues are embedded in paraffin. We also demonstrate immunofluorescence staining of glycan epitopes presented on mucins using plant lectins. The advantage of this approach is that it does not require the use of special fixatives and allows utilizing frozen tissues that may already be preserved in the laboratory.

Unfixed frozen tissue samples embedded in Optimal Cutting Temperature medium (OCT) can be used to study natural distribution and glycosylation of secreted mucus. In this approach tissue processing is minimal and the natural presentation of glycolipids, mucins and glycan-epitopes is preserved. Tissue sections can be analyzed by immunohistochemistry using fluorescence or chromogenic detection.

天然ムチンの分布を調べ不定、凍結組織を用いた

Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight1-3. Secreted mucins, produced by ...

生物学

Induction of Drug-Induced, Autoimmune Hepatitis in BALB/c Mice for the Study of Its Pathogenic Mechanisms

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients with autoimmune hepatitis. The overwhelming majority of patients with DIH are women. The underlying mechanisms of these sex differences in prevalence are unclear because of the paucity of animal models that mimic human disease. Even so, underlying mechanisms are widely believed to be associated with human leukocyte antigen haplotypes and sex hormones. In contrast, using a DIH mouse model, we have uncovered that IL-4 initiated&#160;CD4+ T cells directed against an epitope of cytochrome P450 2E1 induces influx of neutrophils, macrophages and mast cells into the livers of female BALB/c mice. Using this model, we have also shown that IL-33-induced FoxP3+regulatory T cells confer protection against DIH in female and male mice. This DIH model is induced by immunizing mice with an epitope of CYP2E1 that has been covalently altered with a drug metabolite that has been associated with DIH. This epitope is recognized by patients with DIH. Our method induces robust and reproducible hepatitis and autoantibodies that can be utilized to study the pathogenesis of DIH. While in vivo studies can cause undue pain and distress in mice when done improperly, the advantage of an in vivo model is the ability to evaluate the pathogenesis of disease in a large number of mice. Additionally, biological effects of the altered liver proteins can be studied using invasive procedures. The addition of in vitro studies to the experimental design allows rapid repetition and mechanistic analysis at a cellular level. Thus, we will demonstrate our model protocol and how it can be utilized to study in vivo and in vitro mechanisms of DIH.

We describe an in vivo immunization, translational hepatitis model in BALB/c mice that can be utilized to study the pathogenesis of drug-induced autoimmune hepatitis including sex differences seen in this disease. We will describe how this model demonstrates reproducible analyses using in vivo and in vitro experimental techniques.

BALB/cマウスにおける薬物誘発性自己免疫性肝炎の誘導による発症機序の研究

Drug-induced autoimmune hepatitis (DIH) is the most common hepatic drug-induced hypersensitization process observed in approximately 9 to 12% of patients ...

Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. Meningococcus is a transient colonizer of human nasopharynx of approximately 10% of healthy subject. In particular circumstances, it acquires an invasive ability to penetrate the mucosal barrier and invades the bloodstream causing septicaemia. In the latest case, fulminating sepsis could arise even without the consequent development of meningitis. Conversely, bacteria could poorly multiply in the bloodstream, cross the blood brain barrier, reach the central nervous system, leading to fulminant meningitis. The murine models of bacterial meningitis represent a useful tool to investigate the host-pathogen interactions and to analyze the pathogenetic mechanisms responsible for this lethal disease. Although, several experimental model systems have been evaluated over the last decades, none of these were able to reproduce the characteristic pathological events of meningococcal disease. In this experimental protocol, we describe a detailed procedure for the induction of meningococcal meningitis in a mouse model based on the intracisternal inoculation of bacteria. The peculiar signs of human meningitis were recorded in the murine host through the assessment of clinical parameters (e.g., temperature, body weight), evaluation of survival rate, microbiological analysis and histological examination of brain injury. When using intracisternal (i.cist.) inoculum, meningococci complete delivery directly into cisterna magna, leading to a very efficient meningococcal replication in the brain tissue. A 1,000-fold increase of viable count of bacteria is observed in about 18 h. Moreover, meningococci are also found in the spleen, and liver of infected mice, suggesting that the liver may represent a target organ for meningococcal replication.

Here, we describe a method to induce meningococcal meningitis through an intracisternal route of infection in adult mice. We present a step by step protocol of meningococcal infection from the preparation of inoculum to the intracisternal infection; then record the animal survival and evaluate the bacterial loads in murine tissues.

インティリカレルナルデリバリーによるマウスにおける髄膜炎球炎血清群Cの誘導

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

マウス乳腺の凍結切片の間接免疫

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

<meta charset="utf8"></head><body>マウスの授乳研究における牛乳収集のための最適化された一人用法

An Efficient Single-Person Technique for Milk Sampling from Laboratory Mice

Rodent models have been widely used in biology since the early 20th century to study basic physiology and biochemical mechanisms. In lactation and neonatal development research, there is a need to sample milk to determine the composition and how milk may be affected by experimental manipulations, thereby impacting newborn development. Collecting biofluids in sufficient amounts from small rodents can be difficult. Several studies have demonstrated different techniques to obtain milk from mice. However, these techniques often require at least two trained personnel, which may be challenging in some cases.&#160;
Here, we demonstrate a modified milk sampling technique based on a method published in 2009. With this affordable and easy-to-assemble method, one trained person can routinely obtain 0.1&#8211;0.5 mL of milk from a dam under anesthesia in less than 10 min. Using this method, we collected sufficient milk on post-delivery day 2 (PD2) and post-delivery day 10 (PD10). We measured milk macronutrient components and compared them with the existing literature to validate our collection method.&#160;
We recorded that on PD2, milk protein averaged at 70.1 &#177; 5.2 g/L, milk fat was 174.4 &#177; 7.1 g/L, and milk lactose was 12.3 &#177; 0.6 g/L. On PD10, while milk lactose and protein remained at similar concentrations as on PD2, milk fat was significantly higher (224.4 &#177; 17.1 g/L). We also observed that the relative abundance of individual milk proteins varied between PD2 and PD10. More specifically, &#945;S1-casein and whey acidic protein were higher, while &#946;-casein was lower at PD10 compared with PD2. Overall, we demonstrate an efficient single-person technique for milk sampling from mice using a device that can easily be assembled with commonly used laboratory equipment.

<meta charset="utf8"></head><body>実験用マウスからの牛乳サンプリングのための効率的な一人用技術

This article describes how to assemble a device that can be used to collect milk from laboratory mice. This device is affordable, portable, and allows a collection of 0.1&#8211;0.5 mL of milk by one trained person. A detailed milking protocol is also included.&#160;

野生型および肝臓の肝臓試料および牛乳試料中のシアル酸の定量

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