著者らは、首長、首長と蚊フィールドサンプルコレクション中に彼らの協力unwaveing​​用抹茶の社会の世話になっています。博士ダグ·ノリスとリベカケントは塩析プロトコルに非常に貴重な専門知識を提供した。この作品は、ジョンズホプキンスマラリア研究所パイロットグラントシステムによって賄われていた。蚊や寄生虫のDNA実験室の基準は親切にMR4、アメリカン·タイプ·カルチャー·コレクションから寄贈された。

1。モスキート試料の準備解剖<ol><li>顕微鏡を解剖にパラフィルムとマウントの小さなカットの上に置いて蚊。 </li><li>組織を柔らかくするために、脱イオン水の滴でカバー。 </li><li>こうして腹部セクションから頭部と胸部をオフニッキング、胸部と腹部の間の接合部に正確に蚊の死骸を切開。 </li><li>蚊ID番号の詳細や適切ボディ部を（;頭部と胸部セクションの&quot;HT&quot;中腸セクション用などの&quot;m&quot;）を示すようにマークと標識したクリーンなオートクレーブ滅菌1.5 mlマイクロチューブに各解剖蚊のセクションを転送します。 </li><li>パラフィルムを破棄して、慎重に次の蚊を処理する前に70％アルコールで湿らせたキムワイプで顕微鏡ステージを拭いてください。 </li></ol> 2。モスキート検体からのDNA抽出<ol><li>サンプルチューブに脱イオン水をピペットを20μlと国連に沈め蚊部を研削するためにピペットチップを使用iformサスペンション。 </li><li>穏やかな瞬間的なボルテックスでサンプルホモジネートとのミックスにオートクレーブ滅菌1×PBS / 1％サポニン液100μlを加える。 </li><li> 20分間室温でインキュベートする。 </li><li> 2分間20,000 xgで遠心し、上清を捨てる。 </li><li> 100μlの1X PBSでペレットを再懸濁します。 </li><li> 2分間20,000 xgで再び遠心分離し、上清を捨てる。 </li><li>穏やかにボルテックス（5秒）、75μlの滅菌脱イオン水でペレットを再懸濁し、そして脱イオン水で20％（w / v）のChelex-100樹脂の懸濁液の25μlによって。 </li><li>ブンゼンバーナーの炎にあて滅菌23G注射針を使用して、サンプルチューブの蓋にピアスの穴。 </li><li> 10分間フローティングラックに水槽（または加熱ブロック内）の試料懸濁液を沸騰させます。 </li><li> PCRアプリケーションのテンプレートとして使用するための標識したストレージバイアルに1分と転送続くDNA溶液のため、20,000 xgで遠心分離します。 </li></ol> 3。熱帯熱マラリア原虫とハマダラカジェノ属。分子的同定<ol><li> ハマダラカ種10を特定し、遺伝子型Pに、DNAの質11をチェックするために、25μlのPCR反応においてChelex DNA抽出物2.5μlを加え熱帯熱マラリア原虫 12中腸及び唾液腺の感染症。 </li><li>特にPのアンプリコンの収量を最大にするには熱帯熱マラリア原虫の検出は、PCRアッセイで（BSA）は1.5Xウシ血清アルブミンが含まれています。下記の反応組成物をお勧めします（2.5μlのテンプレート、0.25μMプライマー、1.5μMの塩化マグネシウム、200μMのdNTP、1×PCRバッファー、1.5XのBSAおよび1.0U Taq DNAポリメラーゼ、25μlの巻）。 </li><li>抽出物中のDNAの品質をチェックするために、節足動物ミトコンドリアニコチンアミドアデニンジヌクレオチドデヒドロゲナーゼ（NADH）サブユニット4（ND4）遺伝子（プライマーND4FW [5&#39;-GTD YAT TTA TGA TTR CCT​​ AA-3 &#39;]とND4RV [5&#39;の領域を増幅CGD-CTT CTT CCW ADW CGT TC-3 &#39;];製品400bp）11,13を期待しています 。 </li><li> ハマダラカガンビアを区別する電子SL。同胞種（An.ハマダラカssと。arabiensisのみ）は、遺伝子間スペーサー（IGS）領域、（のSNPに隣接する領域を増幅するプライマー国連[5&#39;-GTGTGCCCCTTCCTCGATGT-3 &#39;]、ジョージア州[5&#39;-CTGGTTTGGTCGGCACGTTT- 3 &#39;]、AR [5&#39;-AAGTGTCCTTCTCCATCCTA-3&#39;];製品390bpを期待ハマダラカSS、315bp arabiensis）10,11。 </li><li>タイピングPの熱帯熱マラリア原虫葉酸の薬剤耐性遺伝子多型は、寄生虫ジヒドロ葉酸還元酵素（DHFR）遺伝子のアミノ酸コドン108、51、59、16、164に隣接する領域を増幅したnested PCRを行います。 一次ラウンドプライマー ：M1 [5&#39;-TTTATGATGGAACAAGTCTGC-3 &#39;]とM5 [5&#39;-AGTATATACATCGCTAACAGA-3&#39;] 二次ラウンドプライマー：M3 [5&#39;-TTTATGATGGAACAAGTCTGCGACGTT-3 &#39;] F /で[5&#39;-AAATTCTTGATAAACAACGGAAACCTTTTA-3&#39;] または F [5&#39;-GAAATGTAATTCCCTAGATATGGAATATT-3] M4 [5&#39;-TTAATTTCCCAAGTAAAACTATTAGAGCTTC-3 &#39;]を使用）。 また、アミノ酸のコドン436、437、540、581を含む領域を増幅Pの中にND 613 熱帯熱マラリア原虫 dihydropteroateシンセターゼ（DHPS）遺伝子： 一次ラウンドプライマー R2は[5&#39;-AACCTAAACGTGCTGTTCAA-3 &#39;] R /と[5&#39;-AATTGTGTGATTTGTCCACAA3&#39;]; 二次ラウンドプライマー J：[5&#39;-&#39;TGCTAGTGTTATAGATATAGGTGGAGAAAGC-3&#39;] K / [5&#39;-CTATAACGAGGTATTGCATTTATTGCAAGAA-3 &#39;]と または K：K /で[5&#39;-TGCTAGTGTTATAGATATAGGATGAGCATC-3 &#39;]、 または L [5&#39;-ATAGGATACTATTTGATATTGATATTGGACCAGGATTCG-3 &#39;] L / [5&#39;-5TATTACAACATTTTGATCATTCGCGCAACCGG-3&#39;] 12。 </li><li>製造元の指示に従って30μlの反応における対立遺伝子特異的な制限酵素消化に従うことを条件として、4μlのアンプリコンは、葉酸拮抗剤耐性関連遺伝子多型12を検出します。 </li><li>テーマ5は、電気泳動にエチジウムブロマイド染色した2％アガロースゲルのレーンあたりPCRアンプリコンの液（または制限消化の15μl）（100 - 120V）およびUV透視下でバンドを可視化。 </li></ol>

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特別な利害関係は宣言されません。

ここに提示され単純化されたChelex法は品質ハマダラカ属およびPの抽出を可能にする多様なPCRアプリケーションに従順蚊検体から熱帯熱マラリア原虫の DNA。この手法は、薬剤耐性Pの分子のマラリア媒介蚊の同定および監視のために使用することができる国家マラリア対策プログラムの蚊の原虫対立。簡略化されたChelex技術の利点は、シンプルさ、より少ない試薬および塩析法10（2時間47分、一晩ステップ、 表1）などの標準プロトコルより故にコスト、安全性、短い処理時間（37分）が含まれています。現在の標準プロトコルと比較して、前述の利点と最小限の試薬 ​​の要件（3試薬）（10試薬は、 表1）11,15 は 、一定の試薬 ​​風土病国の研究所に特に優しい簡素Chelexプロトコルを作るサプライチェーンは、多くの場合、維持することは困難です。メソッドの制限は、標準プロトコルのように、それはまた蚊外皮16で発生することが知られているPCR阻害物質の支配下にあったということです。しかし、これは容易にアッセイにおけるBSAを含めることによって緩和される。 BSAはまた首尾よく他のPCRアプリケーション17,18におけるインヒビターに対する増幅エンハンサーとして採用されている。

<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> Chelex-100、50から100ドライメッシュ</td><td> BioRad社</td><td> ＃143-2832 </td><td></td></tr><tr><td>サポニン</td><td> SIGMA </td><td> 142-7425 </td><td></td></tr><tr><td> BSA </td><td>ニューイングランドバイオラボ</td><td> B9001S </td><td></td></tr></tbody></table>

a simple chelex protocol for dna extraction from anopheles spp.

流行国は、ますます彼らの制御プログラム1,2,3,4,5の不可欠な部分として、マラリア原虫と媒介蚊に対する効果的なタイピング、識別および監視のための分子ツールを採用しています。けちな試薬や機器の要件を持つこれらのマラリア制御と排除の取り組みを強化するためのオペレーションズリサーチの正確なアプローチ、シンプルで手頃な価格の方法の確立のために持続可能な6,7,8不可欠です。ここでは、フィールド収集蚊の標本からマラリア原虫とベクターDNAを抽出するためのシンプルなChelexベースの手法を提案する。 私たちは、形態学的に72 ハマダラカSLを同定した。除虫菊スプレーによってキャプチャ156蚊から抹茶でマラリア研究所の2000キロ2近傍内の世帯の部屋を寝にキャッチします。全72 ハマダラカGAMの腹部から頭部と胸部を分離するために解剖した後biae SL。蚊は、2つのセクションが個別に1.5 mlのマイクロチューブに入れ、脱イオン水20μlに浸漬した。滅菌ピペットチップを用いて、各蚊セクションは別々に脱イオン水で均一な懸濁液に均質化した。他の10μlを別オートクレーブした1.5​​ mlチューブに移しつつ、各蚊セクションから続くホモジネートを、10μlを保持した。別のアリコートを簡略Chelexまたは抽出プロトコル9,10塩析標準のいずれかの方法でDNA抽出を行った。それはDNA抽出9時の蛋白質沈殿工程のために有害な有機溶媒（例えば、フェノール、クロロホルムなど）の代わりに高い塩濃度を採用しているため、塩析プロトコルは、いわゆる、広く使われています。 抽出物は、節足動物ミトコンドリアニコチンアミドアデニンジヌクレオチドdehydrogeを標的としたプライマーを用いたPCR増幅のための鋳型として用いたnase（NADH）サブユニット4遺伝子（ND4）は、 熱帯熱マラリア原虫（Plasmodium falciparum）感染の 12のタイピングのためのDNAの質11、 ハマダラカの同胞種10の識別のためのPCRとnested PCR法を確認することができます。 DNAの品質（ND4）を使用して比較すると、PCRが確立された塩析プロトコルに対する相対Chelexアプローチのための93％の感度と82％の特異性を示した。感度と特異度の対応する値は Pのために、それぞれの兄弟種同定、PCRと92％と80％を使用して、それぞれ100％と78％であった熱帯熱マラリア原虫検出PCR。 Chelexまたはすべての3つのPCRアプリケーション間でプロトコルを塩析レギュラーとリコン信号を与える検体の割合に有意差は認められなかった。 Chelexアプローチは、3つの簡単な試薬、完了までに37分を要し、一方、一晩工程を含む、10種類の試薬および2時間と47分 &#39;処理時間を伴プロトコルを塩析。我々の結果は、番目を表示Chelex方法で、既存の塩析、抽出に匹敵し、一定の試薬のサプライチェーンは頻繁に維持することが困難である資源の限られた設定で、シンプルかつ持続可能なアプローチとして代用できます。

蚊のDNA抽出質（ 図1）、 ハマダラカarabiensis分子の同定（ 図2）とPのためのPCRアッセイからの結果の例簡略化されたChelex法ははるかに少ない手順（ 表1）にもかかわらず、プロトコル10を塩析標準と同様の結果が得られることを熱帯熱検出（ 図3）を表示します。それぞれの抽出液に匹敵するDNAの品質で、それは驚くべきことではないことに対するサンプル陽性率。ハマダラカ同胞種だけでなく、寄生虫感染率はマクネマーのカイ二乗検定（ 図3）に基づいて、統計的に差は認められなかった。 感度（％）は次のように計算されたTP /（TP + FN）TPは真の陽性を示し、FN​​は偽陰性を示している* 100。特異性（％）は、TN /のように決定したTNは真の陰性を示し、FPは偽陽性を示し（TN + FP）* 100、。 DNAの品質（ND4）PCRは、設立されるようにプロトコルを塩析金本位制に比べChelexアプローチのための93％の感度と82％の特異性を示した。感度と特異度の対応する値は Pのために、それぞれの兄弟種同定、PCRと92％と80％を使用して、それぞれ100％と78％であった熱帯熱マラリア原虫検出PCR。 反応混合物にBSAの添加は、簡略化さChelexと正規両方のプロトコルを塩析するため、PCR阻害物質による14の救済へのPCR陽性（ 図3）の一般的な増加をもたらした。しかし、この増加はChelex抽出した（p = 0.039）上のDNAの質（ND4 PCR）を除いて統計的に有意ではなかった。 P上の対立遺伝子特異的制限酵素消化熱帯熱マラリア DHFRは（または他の標的遺伝子）アンプリコンは、薬剤抵抗性対立遺伝子（;唾液腺のデータは示されていない図4）の中腸及び唾液腺マラリア感染症の遺伝子タイピングを可能にします。 <table border = &quot;1&quot;&gt; <tbody><tr><td colspan="2"> シンプルChelex手順 </td><td colspan="2"> 標準では、手順を塩析 </td></tr><tr><td> 手順 </td><td> 試薬 </td><td> 手順 </td><td> 試薬 </td></tr><tr><td><ol><li> 1.5 mlのマイクロチューブに検体を追加し、1×PBS / 1％サポニン溶液（2分）100μlにホモジナイズする。 </li><li> 20分間室温で残す。 </li><li> 2分間20,000 xgでスピンし、上清を捨てる。 </li><li> 1X100μlのPBSを加え、一瞬（3秒）ボルテックスで静かに混和する。 </li><li> 2分間20,000 xgでスピンし、上清を捨てる。 </li><li>脱イオン水および75μlの滅菌脱イオン水で20％（w / v）のChelex25μlを追加します。 </li><li> 10分間ホット、無菌の23G注射針、沸騰管内容物を使用して、サンプルチューブの蓋にピアスの穴。 </li><li> 2で微量遠心チューブをスピン1分間0000 xgで。 </li><li>使用するまで-20℃（25μlのPCR反応液に2.5μL）で標識し、無菌バイアルと店舗DNA溶液に上清を移してください。 </li></ol></td><td> PBS サポニン Chelex-100ビーズ</td><td><ol><li> 1.5 mlのマイクロチューブに検体を追加し、1％のDEPC（2分）で100μlベンダーバッファー*でホモジナイズする。 </li><li> 1時間65℃でインキュベートする。 </li><li> 15μlの冷8Mの酢酸カリウムを追加します。穏やかに混合し、45分間氷上でインキュベートする。 </li><li> 10分と新しい1.5 mlチューブに上清を転送するために20,000 xgでマイクロチューブにサンプルをスピン。 </li><li> 100％エタノールを250μlを加え、チューブを反転してよく混ぜる。 </li><li>室温で5分間静置して下さい。 </li><li> 15分間20,000 xgでサンプルをスピン。 </li><li>吸引し、上清を捨て、ペレットがチューブ（30メートルで完全に乾かし残し）インチ</li><li> 4℃で一晩0.1×SSC +アーゼ（10μg/ ml）を20μlにペレットを再懸濁し℃、 </li><li>使用するまで-20℃で80μlのDEPC水とストアを追加します。 </li></ol></td><td>ジエチル（DEPC） *ベンダーバッファ： 0.1MのNaCl 0.2 Mショ糖 0.1Mトリス-HCl 0.05 M EDTA、pH9.1の DEPC水で0.5％のSDS 8 M酢酸カリウムエタノール 0.1X食塩クエン酸ナトリウム（SSC）は、バッファリボヌクレアーゼ（10μg/ ml）を</td></tr><tr><td colspan="2">合計時間：37分</td><td colspan="2">合計時間：2時間47分、プラス一晩</td></tr></tbody></table> 表1。シンプルChelexプロトコル用試薬および時間要件のステップ比較によりステップとスタンドARDは蚊の標本からDNAを抽出するための方法を塩析。 <img alt="図1" src="/files/ftp_upload/3281/3281fig1.jpg" /> ハマダラカarabiensisフィールドサンプルの&quot;M&quot;の抽出物を塩析簡略化Chelex &quot;C&quot;と標準からのDNAの質の1。ND4ミトコンドリアPCRの比較図 。ノースカロライナ、ネガティブコントロール、M1とC1、C2、M2と、M3とC3、およびM4とC4は、塩析と同じからChelex抽出対になっています。 arabiensisの蚊の標本。 L、100 bpのDNAラダー。 <img alt="図2" src="/files/ftp_upload/3281/3281fig2.jpg" /> 。。ハマダラカarabiensis C1とM1、C2とM2は、C3とM3、C4およびM4は同じ蚊の標本のために&quot;M&quot;と簡略化しChelex &quot;C&quot;のDNA抽出物を塩析からそれぞれのアンプリコンを示すために、図2分子の同定、PCR、AR、 ハマダラカarabiensisポジティブコントロール、L、100 bpのDNAラダー。 <img alt="図3" src="/files/ftp_upload/3281/3281fig3.jpg" /> 図3 Chelexとハマダラカarabiensis（AR）10、節足動物NADHデヒドロゲナーゼ遺伝子4（ND4）DNAの品質テスト11、葉酸薬剤耐性Pの分子同定のためのPCRアッセイと、蚊フィールドサンプルのDNA抽出、塩析で陽性の検出。熱帯熱マラリア DHFR遺伝子タイピング12（F-M4）。反応混合物中のBSAの有無にかかわらず実行するアッセイのために示された結果。マクネマーのカイ二乗検定は、DNAからポジティブPCRのパーセントの違いが簡素Chelexとプロトコルを統計学的に有意であった塩析標準から抽出されたかどうかを判断するために使用されていました。 <img alt="図4" src="/files/ftp_upload/3281/3281fig4.jpg" /> <stroNG&gt;図4：タイピングPに簡略化されたChelexプロトコルの応用薬剤抵抗性対立遺伝子のために蚊の原虫感染症（中腸のデータが示されている）。 Pのコドン108に隣接するリコンにBstN私は消化熱帯熱マラリア DHFR遺伝子12は蚊サンプル（;半ば腸データは示さM1〜M5）でcycloguanil耐性S108T変異体を示しています。 U、未消化の522 bpのアンプリコン; FCR3、実験室標準P. S108Tを運んで熱帯熱マラリア原虫陽性対照クローン、K1、P.熱帯熱マラリア原虫実験室標準cycloguanilセンシティブS108Nを保有するクローンネガティブコントロール、L、100 bpのDNAラダー。

Watch this Scientific Journal Video about A Simple Chelex Protocol for DNA Extraction from Anopheles spp. at JoVE.com

ハマダラカ属から DNA を抽出するための簡単な Chelex プロトコル|免疫学と感染症 |ビデオ

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

蚊の標本から品質マラリア原虫とベクターDNAを抽出するために迅速かつ現実的な方法が記載されている。 Chelex樹脂のキレート特性を生かし、簡単な方法は、マラリア蚊の中腸及び唾液腺の段階で寄生虫と同様の分子同定の遺伝子型が可能<em&gt;ハマダラカ</emPCRによる&gt;同胞種。

からのDNA抽出のためのシンプルなChelexプロトコル<em&gt;ハマダラカ</em&gt;属。

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

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A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

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54.8K ビュー.  Malaria Institute at Macha. 蚊の標本から品質マラリア原虫とベクターDNAを抽出するために迅速かつ現実的な方法が記載されている。 Chelex樹脂のキレート特性を生かし、簡単な方法は、マラリア蚊の中腸及び唾液腺の段階で寄生虫と同様の分子同定の遺伝子型が可能ハマダラカ同胞種。

ビデオ: A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

Mosquitoes are vectors for a number of disease-causing pathogens such as the yellow fever virus, malaria parasites and filarial worms. Laboratories are investigating anti-pathogen components of the innate immune system in disease vector species in the hopes of generating transgenic mosquitoes that are refractory to such pathogens1, 2. The innate immune system of mosquitoes consists of several lines of defense 3. Pathogens that manage to escape the barrier imposed by the epithelium-lined mosquito midgut 4 enter the hemolymph and encounter circulating hemocytes, important cellular components that encapsulate and engulf pathogens 5, 6. Researchers have not found evidence for hematopoietic tissues in mosquitoes and current evidence suggests that the number of hemocytes is fixed at adult emergence and numbers may actually decline as the mosquito ages 7. The ability to properly collect and identify hemocytes from medically important insects is an essential step for studies in cellular immunity. However, the small size of mosquitoes and the limited volume of hemolymph pose a challenge to collecting immune cells. 
Two established methods for collecting mosquito hemocytes include expulsion of hemolymph from a cut proboscis 8, and volume displacement (perfusion), in which saline is injected into the membranous necklike region between the head and thorax (i.e., cervix) and the perfused hemolymph is collected from a torn opening in a distal region of the abdomen 9, 10. These techniques, however, are limited by low recovery of hemocytes and possible contamination by fat body cells, respectively 11. More recently a method referred to as high injection/recovery improved recovery of immunocytes by use of anticoagulant buffers while reducing levels of contaminating scales and internal tissues 11. While that method allows for an improved method of collecting and maintaining hemocytes for primary culture, it entails a number of injection and collecting steps that are not necessary if the downstream goal is to collect, fix and stain hemocytes for diagnostics. Here, we demonstrate our method of collecting mosquito hemolymph that combines the simplicity of perfusion, using anticoagulant buffers in place of saline solution, with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes.

A Protocol for Collecting and Staining Hemocytes from the Yellow Fever Mosquito Aedes aegypti

A simplified yet accurate method to collect and stain mosquito hemocytes is described. Our method combines the simplicity of perfusion with the accuracy of high injection techniques to isolate clean preparations of hemocytes in Aedes mosquitoes. This method facilitates studies requiring knowledge of the types of hemocytes and their abundance.

黄熱病の蚊から血球を収集し、染色のためのプロトコルネッタイシマカ</em

 Mosquitoes are vectors for a number  of disease-causing pathogens such as the yellow fever virus, malaria parasites  and filarial worms. Laboratories are ...

免疫・感染学

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol (PEG) Precipitation

Hens can be immunized by means of i.m. vaccination (Musculus pectoralis, left and right, injection volume 0.5-1.0 ml) or by means of Gene-Gun plasmid-immunization. Dependent on the immunogenicity of the antigen, high antibody-titres (up to 1:100,000 - 1:1,000,000) can be achieved after only one or 3 - 4 boost immunizations. Normally, a hen lays eggs continuously for about 72 weeks, thereafter the laying capacity decreases. This protocol describes the extraction of total IgY from egg yolk by means of a precipitation procedure (PEG. Polson et al. 1980). The method involves two important steps. The first one is the removal of lipids and the second is the precipitation of total IgY from the supernatant of step one. After dialysis against a buffer (normally PBS) the IgY-extract can be stored at -20&deg;C for more than a year. The purity of the extract is around 80 %, the total IgY per egg varies from 40-80 mg, dependent on the age of the laying hen. The total IgY content increases with the age of the hen from around 40 mg/egg up to 80 mg/egg (concerning PEG precipitation). The laying capacity of a hen per year is around 325 eggs. That means a total potential harvest of 20 g total IgY/year based on a mean IgY content of 60 mg total IgY/egg (see Table 1).

IgY Technology: Extraction of Chicken Antibodies from Egg Yolk by Polyethylene Glycol PEG Precipitation

This protocol describes in particular the extraction of total IgY from egg yolk by means of polyethylene glycol precipitation and gives general information about IgY technology.

IGY技術：ポリエチレングリコール（PEG）沈殿により卵黄からニワトリ抗体の抽出

Hens can be immunized by means of  i.m. vaccination (Musculus pectoralis, left and right, injection volume  0.5-1.0 ml) or by means of Gene-Gun ...

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important plant parasitic nematodes. The microscopic second-stage juvenile (J2), molted once in the egg, is the infective stage. The J2s hatch from the eggs, move freely in the soil within a film of water, and locate root tips of suitable plant species. After penetrating the plant root, they migrate towards the vascular cylinder where they establish a feeding site and initiate feeding using their stylets. The multicellular feeding site is comprised of several enlarged multinuclear cells called 'giant cells' which are formed from cells that underwent karyokinesis (repeated mitosis) without cytokinesis. Neighboring pericycle cells divide and enlarge in size giving rise to a typical gall or root knot, the characteristic symptom of root-knot nematode infection. Once feeding is initiated, J2s become sedentary and undergo three additional molts to become adults. The adult female lays 150-250 eggs in a gelatinous matrix on or below the surface of the root. From the eggs new infective J2s hatch and start a new cycle. The root-knot nematode life cycle is completed in 4-6 weeks at 26-28&deg;C.
Here we present the traditional protocol to infect plants, grown in pots, with root-knot nematodes and two methods for high-throughput assays. The first high-throughput method is used for plants with small seeds such as tomato while the second is for plants with large seeds such as cowpea and common bean. Large seeds support extended seedling growth with minimal nutrient supplement. The first high throughput assay utilizes seedlings grown in sand in trays while in the second assay plants are grown in pouches in the absence of soil. The seedling growth pouch is made of a 15.5 x 12.5cm paper wick, folded at the top to form a 2-cm-deep trough in which the seed or seedling is placed. The paper wick is contained inside a transparent plastic pouch. These growth pouches allow direct observation of nematode infection symptoms, galling of roots and egg mass production, under the surface of a transparent pouch. Both methods allow the use of the screened plants, after phenotyping, for crossing or seed production. An additional advantage of the use of growth pouches is the small space requirement because pouches are stored in plastic hanging folders arranged in racks.

High and Low Throughput Screens with Root-knot Nematodes Meloidogyne spp.

Two distinct methods to screen plants with root-knot nematodes are described. The described approaches include high-throughput screens with nematodes in a nondestructive manner facilitating the use of these plants in breeding programs.

根こぶ線虫、高および低スループット画面サツマイモ属。</em

Root-knot nematodes (genus Meloidogyne) are obligate plant parasites. They are extremely polyphagous and considered one of the most economically important ...

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules 1. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes 2 and restrict their environment to a more favorable low oxygen pressure 3. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.
Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria 4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor 6. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)7,8 has also been associated with severe disease 9.
One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps 10. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM 11. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian 12 and Mozambican patients 13, (although not in Malian 14).
With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay 15. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

This method investigates the platelet-mediated clumping phenotype of Plasmodium falciparum-infected erythrocytes (pRBC) in clinical isolates. This is performed by isolating and co-incubating platelet-rich plasma and a suspension of pRBC.

の血小板媒介クランピングのためのシンプルなプロトコル<em&gt;熱帯熱マラリア原虫</emリソース悪い設定で、&gt;感染した赤血球

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of the intracellular protein levels of bacterial species is of importance to understanding the pathogenic mechanisms of diseases caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

全タンパク質の抽出および2次元ゲル電気泳動法のための<em&gt;バークホルデリア</em&gt;種

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped RNA virus belonging to the family Flaviviridae. Current treatment is not fully effective and causes adverse side effects. There is no HCV vaccine available. Thus, continued effort is required for developing a vaccine and better therapy. An HCV cell culture system is critical for studying various stages of HCV growth including viral entry, genome replication, packaging, and egress. In the current procedure presented, we used a wild-type intragenotype 2a chimeric virus, FNX-HCV, and a recombinant FNX-Rluc virus carrying a Renilla luciferase reporter gene to study the virus replication. A human hepatoma cell line (Huh-7 based) was used for transfection of in vitro transcribed HCV genomic RNAs. Cell-free culture supernatants, protein lysates and total RNA were harvested at various time points post-transfection to assess HCV growth. HCV genome replication status was evaluated by quantitative RT-PCR and visualizing the presence of HCV double-stranded RNA. The HCV protein expression was verified by Western blot and immunofluorescence assays using antibodies specific for HCV NS3 and NS5A proteins. HCV RNA transfected cells released infectious particles into culture supernatant and the viral titer was measured. Luciferase assays were utilized to assess the replication level and infectivity of reporter HCV. In conclusion, we present various virological assays for characterizing different stages of the HCV replication cycle.

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

C型肝炎ウイルスの複製を分析するためのプロトコル

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

簡単かつ迅速なプロトコル非酵素的に浸潤リンパ球の分析のための新鮮なヒト組織を解離

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Efficient Isolation Protocol for B and T Lymphocytes from Human Palatine Tonsils

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to the palatine tonsils situated at the lateral walls of the oral part of the pharynx. Surgically removed palatine tonsils provide a convenient accessible source of B and T lymphocytes to study the interplay between foreign pathogens and the host immune system. This video protocol describes the dissection and processing of surgically removed human palatine tonsils, followed by the isolation of the individual B and T cell populations from the same tissue sample. We present a method, which efficiently separates tonsillar B and T lymphocytes using an antibody-dependent affinity protocol. Further, we use the method to demonstrate that human adenovirus infects specifically the tonsillar T cell fraction. The established protocol is generally applicable to efficiently and rapidly isolate tonsillar B and T cell populations to study the role of different types of pathogens in tonsillar immune responses.

Palatine tonsils are a rich source of B and T lymphocytes. Here we provide an easy, efficient and rapid protocol to isolate B and T lymphocytes from human palatine tonsils. The method described has been specifically adapted for studies of the viral etiology of tonsil inflammation known as tonsillitis.

ヒトパラティーノ扁桃腺からBおよびTリンパ球の効率的な単離プロトコール

Tonsils form a part of the immune system providing the first line of defense against inhaled pathogens. Usually the term &#8220;tonsils&#8221; refers to ...

A Simple and Efficient Approach to Construct Mutant Vaccinia Virus Vectors

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been successfully employed for editing genomes of various organisms. Here we describe a protocol for editing the vaccinia virus (VV) genome in the cytoplasm of VV-infected CV-1 cells using the RNA-guided Cas9. RNA-guided Cas9 induces double-stranded DNA breaks facilitating homologous recombination efficiently and specifically in the targeted site of VV and a transgene can be incorporated into these sites by homologous recombination. By using a site-specific homologous vector with transgene(s), a N1L gene-deleted VV with the red fluorescence protein (RFP) gene incorporated in this region was generated with a successful recombination efficiency 10 times greater than that obtained from the conventional homologous recombination method. This protocol demonstrates successful use of RNA-guided Cas9 system to generate mutant VVs with enhanced efficiency.

Vaccinia virus (VV) has been widely used in biomedical research and the improvement of human health. This article describes a simple, highly efficient method to edit the VV genome using a CRISPR-Cas9 system.

突然変異体ワクシニアウイルスベクターを構築するためのシンプルで効率的なアプローチ

The CRISPR-associated endonuclease Cas9 can edit particular genomic loci directed by a single guide RNA (gRNA). The CRISPR/Cas9 system has been ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

からのDNA抽出のためのシンプルなChelexプロトコル

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根こぶ線虫、高および低スループット画面サツマイモ属。

の血小板媒介クランピングのためのシンプルなプロトコル

全タンパク質の抽出および2次元ゲル電気泳動法のための

C型肝炎ウイルスの複製を分析するためのプロトコル

簡単かつ迅速なプロトコル非酵素的に浸潤リンパ球の分析のための新鮮なヒト組織を解離

ヒトパラティーノ扁桃腺からBおよびTリンパ球の効率的な単離プロトコール

突然変異体ワクシニアウイルスベクターを構築するためのシンプルで効率的なアプローチ

関連付けられているウイルスおよび細胞タンパク質の同定のウイルス DNA の精製