我々は、有意義な議論と技術支援のために我々の研究室のメンバーに感謝。このビデオプレゼンテーションは、研究と獣医病理学科のための副学部長、獣医健康科学センター、オクラホマ州立大学によってサポートされていました。研究がMinisterioデCiencia電子Innovación、スペイン（プロジェクトBFU2008-01244/BMC）、JFにCSIC学内プロジェクトPA1002451によって資金を供給された、ウォルターR. SitlingtonはKMK、CVHS 2009 RAC助成金、OAESに食用動物の研究のための椅子を寄附アニマルヘルスファンドと米国農務省、国立研究イニシアチブ競争グラント、第2007〜04613。

1。 dsRNAの世代。 <ol><li> dsRNAのin vitroでの転写および合成のためのT7プロモーター配列を含むオリゴヌクレオチドプライマーを合成する（例えば、 カクマダニ属のためにsubolesinオリゴヌクレオチドプライマーD8AAT75を使用アベマキ ： 5&#39; - TAATACGACTCACTATAGGGTACTGACTGGGATCCCCTGCACAGT - 3&#39;とD8DVT73：5&#39; - TAATACGACTCACTATAGGGTACTCGAGCTTGGTGGAAAGGACG - 3&#39;）。 </li><li> RT - PCRは、それぞれのオリゴヌクレオチドプライマーとダニのトータルRNA 1-10 ngの10 pmolのを使用して標的遺伝子を増幅する。 </li><li> PCR産物を精製する。 </li><li>精製したPCR産物の8μLを用いてdsRNAを合成する。 </li><li>スペクトロメトリーによりdsRNAを定量化する。 </li></ol> 2。 dsRNAを持つマダニの注射。 2.1。注射のためのティックの準備。 <ol><li>最初に、ダニを保持するためにチューブトップを細かくメッシュワイヤースクリーンを通してソリューションをデカント、50mLの使い捨て遠心管に各ソリューションでそれらを振盪することによってソリューションの一連のティックを洗う。洗濯ダニのためのソリューションのシーケンスは、水道水、3％過酸化水素、蒸留水の2回洗浄、70％エタノールおよび蒸留水でさらに2回洗浄です。 </li><li>ペーパータオルの上でダニをブロット乾燥。 </li><li>実験に応じて、20〜50のグループにティックをカウント、実験群の数としっかりフィットふたとラベルを1.25オンスのプラスチック製のコップで各グループからダニを置きます。 </li></ol> 2.2。射出チームティック。 RNAiのチームは3人で構成されています：（1）赤歯科用ワックスのシートに貼り付けられた二重粘着テープ上の各目盛りを置いた一人、後にダニを監視目盛りと（3）一人を注入する（2）一人注射は、それらを有効にダニにCO 2を吹き込みと実験群の番号のラベルが付いてカップに住んでいるダニをカウントします。すべてのチームメンバーは、使い捨ての手袋を着用しなければならない。 2.3。注射のためのダニの配置。 <ol><li>デュモン細かい鉗子を使用してダニをキャプチャし、赤歯科ワックスの3&quot;× 6&quot;シートに貼付ダブル粘着テープの上に腹側を上に置きます。チックは密接に5ティックのグループで一緒に配置されている。 </li><li>さらに、それらを抑制するために、全5ダニの口器にわたってマスキングテープの小片を置きますが、注射のプロセスがダニインジェクタ（図1）によって観察できるように公開されたボディのほとんどを残すこと。 </li></ol> 2.4。ダニの注射。 <ol><li>ダニは、外骨格の腹側表面の右下腹部に注入されます。 </li><li>最初に、ピアス½&quot;、29ゲージの針（図2a）を装備Monojectのインスリン注射器を使用して外骨格に穴。 </li><li> 45 °傾斜したポイントで1インチ、33ゲージの針を使用してカスタムメイドのハミルトンシリンジを用いて- （μLあたり5 × 10 11分子が5 × 10 10）（図2b）のdsRNAのソリューションの0.2から0.5μLで、すぐにチックを注入する。針は、dsRNAの配置と保持を保証するためにもダニの空洞内に配置する必要があります。いくつかの流体は、注射部位（図2c）から脱出する可能性があります。ケアは、以上の血リンパの損失を引き起こすとダニの死を引き起こす可能性ダニを、注入しないように注意してください。 </li><li>各実験群で注射を完了した後にハミルトンシリンジを清掃してください。別の実験群のために使用する前に。 3％の過酸化水素を含むビーカーから最初の注射器をいっぱいにし、廃棄物の容器に追放し、15回繰り返す。滅菌水の入ったビーカーからシリンジを記入し、廃棄物の容器に追放し、15回繰り返す。曲がった場合、プランジャーがスムーズに移動し、ダニの注入に必要な優しいタッチに反応しない、ので、ハミルトンシリンジのプランジャーを曲げないように注意してください。 </li></ol> 2.5。注入後のダニの治療。 <ol><li>細かい鉗子ですぐにダブル粘着テープから注入された目盛りをピックアップし、プラスチックの回収容器（約6&quot;× 6&quot;とダニの脱出を防ぐためにマスキングテープで囲ま）に置きます。ティックは、注射後しばらく非アクティブになりますが、すぐに皿の周りにクロールを開始する必要があります。 </li><li>息がすぐにダニを有効にするための回収容器にそれらを配置した後にダニにCO 2を 。ダニは、クロールとアクティブされると、注射の傷は急速に治癒するが、これはほとんど生き残っていくだろう。 </li><li>各実験群の数に応じて目盛りを数えて、しっかりと取り付けられた蓋のラベルが付いたプラスチック製のコップの中に置いてください。交換用の目盛りは、いずれかが次の実験群を注入する前に死亡したことを置き換えるために注入されるべきである。 </li></ol> 2.6。持株ティック。 <ol><li> ° Cと95％の相対時間22〜25で12時間暗光周期：湿度チャンバ内でダニを置きます（12時間光umidity）と1日間保持する。 </li><li>ダニ送り細胞、実験群に1つずつ配置ダニは、羊に接着し、それらをuninjected雄または雌ダニの数と同じ数（注入されていない方の性別）を供給することができます。たらふく女性チックそのフィードは、給餌の10日後に羊から削除されるか、コントロールの女性がホストを降ろしているときにそれらを収集し、秤量する。 </li><li>カートンでダニを置き、そして産卵の完了まで、湿度チャンバ内に保持する。重み付けして、グループ内のすべてのダニによって生成された卵塊を産卵を評価する。 </li></ol> 2.7。 RNAiの後のダニの表現型の解析。 <ol><li>生き残ったダニの数を決定することによって、餌の後に表現型をチェック評価し、体重、産卵と卵の肥沃度をチェックします。しかし、他の分析は、標的遺伝子と研究の目的に応じて実施することができる。 </li></ol> 3。 RT - PCRによる遺伝子サイレンシングを確認する分析。 <ol><li>授乳後のコントロールを注入し、dsRNAを注入されたグループから個々のダニから唾液腺や内臓を細かく分析。 </li><li>個々の組織サンプルからトータルRNAを抽出する。 </li><li>リアルタイムRT - PCRによって個々の組織における標的遺伝子の転写産物を分析し、ダニの16Sに対してRNAレベルを正常化するには、genNorm法（Bio - Rad社iQ5のStandard Edition、バージョン2.0で実装されたddCT方式）を使用してrRNAの。 </li><li>唯一のアンプリコンが形成されていることを確認するために反応の終了時に、その解離曲線を実行するすべてのサンプルに対して同じ温度範囲で一貫してアンプリコンの変性。 </li><li>制御注入し、学生の`sのt -検定（P = 0.05）を用いて二本鎖RNAを注入されたティック間のmRNAレベル（正規化されたCt値）を比較する。 </li></ol> 4。代表的な結果： ここに記述されているプロトコルは、多くの異なるマダニの種でRNAi実験のための我々の研究室（表1）で使用されています。ティックに注入されたdsRNAの量は、ダニの大きさによって変化します。大きくダニの種は、より大きなボリュームを収容することができます。ネガティブコントロールの目盛りは、無関係のdsRNAを注入する必要があります。このようなsubolesin 14-19,22-25,27-32,34とβ-アクチン20,21のようないくつかのdsRNAはポジティブコントロールとして使用することができる。それは混合のdsRNAソリューションを避けるために、治療の間に注射器を洗浄することが重要であることに注意してください。プロトコルが適切に行われた場合、5％未満の死亡率は、24時間後に注射の手順から入手してください。ダニの遺伝子のノックダウン後の典型的な表現型は防御抗原をチェックするための画面にするためにdsRNAのプールを注入ダニのパネルと図3に示します。 <table border="1"><tbody><tr><td> 種ティック </td><td> dsRNAは、注入 </td><td> 参考文献 </td></tr><tr><td> scapularisマダニ属 </td><td> cDNAライブラリー、subolesin、アクチン、ヌクレオチダーゼ、NF - KB、akirin </td><td> 21、22、29、30 </td></tr><tr><td> カクマダニ属アベマキ </td><td> subolesin、GST、ユビキチン、vATPase、セレンMとW2A、造血幹細胞/前駆細胞アクチン、タンパク質のような26Sプロテアソームサブユニット、ferritin1、varisin、akirin </td><td> 15、19、22、24、26、30から32 </td></tr><tr><td> カクマダニ属marginatus </td><td> subolesin </td><td> 22 </td></tr><tr><td> キララamericanum </td><td> cDNAライブラリー、subolesin、akirin </td><td> 17、22、30 </td></tr><tr><td> キララhebraeum </td><td> subolesin、voraxin </td><td> 28 </td></tr><tr><td> Rhipicephalusのsanguineus </td><td> Rs86、subolesin </td><td> 22、23 </td></tr><tr><td> Rhipicephalus microplus </td><td> GST、ユビキチン、セレン、Bm86、Bm91、subolesin、GI、GIII、EF1a、鞭毛状の糸タンパク質、フォンウィルブランド ファクタ</td><td> 16、18、25、27 </td></tr><tr><td> Rhipicephalusのannulatus </td><td>ユビキチン、subolesin、EF1a、GIII </td><td> 16 </td></tr></tbody></table> 表1：RNAiのプロトコルが使用されている種にチェックを入れます。 <img src="/files/ftp_upload/2474/2474fig1.jpg" alt="図1" /> 図1赤歯科用ワックスのシートに付着ダブル粘着テープの目盛りの配置、上腹側、。ダニは、マスキングテープの小片がインジェクタが注入中にダニの体を観察しながら、さらにダニを確保するために口器上に配置され、その後、5グループに配置されます。 <img src="/files/ftp_upload/2474/2474fig2.jpg" alt="図2" /> 図2。注入手順はインスリンSYとダニの外骨格の右下の象限にピアス（）が含まれています注射部位を作成するために29ゲージの針を備えたringe、（c）のほとんどがダニの血リンパ/いくつかの漏洩につながる33ゲージの針でハミルトンシリンジを使用して、このサイトでのdsRNAの（b）の即時注入流体。 <img src="/files/ftp_upload/2474/2474fig3.jpg" alt="図3" /> 図3。RNAiのはキララamericanumのダニ防御抗原をスクリーニングするために使用されたダニ6つのグループのパネル。ポジティブsubolesin RNAiを制御し、負の無関係なdsRNAをコントロールと比較した場合、ティック単位での表現型の変化を見ることができます。この実験では各グループのダニの死亡率、重量、および産卵に対するRNAiの効果が統計的に解析した。

<ol>
	<li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview:+Ticks+as+vectors+of+pathogens+that+cause+disease+in+humans+and+animals.">Overview: Ticks as vectors of pathogens that cause disease in humans and animals.</a> Front. Biosci. 13, 6938-6946 (2008).</li><li>Peter, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mosquito+control-Lessons+from+the+past,+solutions+for+the+future.">mosquito control-Lessons from the past, solutions for the future.</a> Vet. Parasitol. 132, 205-215 (2005).</li><li>Barker, S. C., Murrell, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematics+and+evolution+of+ticks+with+a+list+of+valid+genus+and+species+names.">Systematics and evolution of ticks with a list of valid genus and species names.</a> Parasitol. 129, S15-S36 (2004).</li><li>Willadsen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tick+control:+Thoughts+on+a+research+agenda.">Tick control: Thoughts on a research agenda.</a> Vet. Parasitol. 138, 161-168 (2006).</li><li>de la Fuente, J., Kocan, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+development+of+vaccines+for+control+of+ixodid+tick+species.">Strategies for development of vaccines for control of ixodid tick species.</a> Parasite Immunol. 28, 275-283 (2006).</li><li>Fire, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+and+specific+genetic+interference+by+double-stranded+RNA+in+Caenorhabditis+elegans.">Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.</a> Nature. 391, 806-811 (1998).</li><li>Cerutti, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference:+traveling+in+the+cell+and+gaining+functions.">RNA interference: traveling in the cell and gaining functions.</a> Trends Genet. 19, 39-46 (2003).</li><li>Kavi, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+silencing+in+Drosophila.">RNA silencing in Drosophila.</a> FEBS Lett. 579, 5940-5949 (2005).</li><li>Mello, C. C., Conte, D. J. r. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revealing+the+world+of+RNA+interference.">Revealing the world of RNA interference.</a> Nature. 431, 338-342 (2004).</li><li>Zhou, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+and+potential+applications.">RNA interference and potential applications.</a> Curr. Top. Med. Chem. 6, 901-911 (2006).</li><li>Ramakrishnan, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+RNA+interference+in+tick+salivary+gland+research.">Application of RNA interference in tick salivary gland research.</a> J. Biomol. Tech. 16, 297-305 (2005).</li><li>Aljamali, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference:+applicability+in+tick+research.">RNA interference: applicability in tick research.</a> Exp. Appl. Acarol. 28, 89-96 (2002).</li><li>Aljamali, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+in+ticks:+a+study+using+histamine+binding+protein+dsRNA+in+the+female+tick+Amblyomma+americanum.">RNA interference in ticks: a study using histamine binding protein dsRNA in the female tick Amblyomma americanum.</a> Insect. Mol. Biol. 12, 299-305 (2003).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+for+the+study+and+genetic+manipulation+of+ticks.">RNA interference for the study and genetic manipulation of ticks.</a> Trends Parasitol. 23, 427-433 (2007).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomic+studies+of+tick+cells+in+response+to+infection+with+the+cattle+pathogen,+Anaplasma+marginale.">Functional genomic studies of tick cells in response to infection with the cattle pathogen, Anaplasma marginale.</a> Genomics. 90, 712-722 (2007).</li><li>Almaz&#228;N, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+Rhipicephalus+(Boophilus)+microplus+candidate+protective+antigens+for+the+control+of+cattle+tick+infestations.">Identification and characterization of Rhipicephalus (Boophilus) microplus candidate protective antigens for the control of cattle tick infestations.</a> Parasitol. Res. 106, 471-479 (2010).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+protective+antigens+by+RNA+interference+for+control+of+the+lone+star+tick,+Amblyomma+americanum.">Identification of protective antigens by RNA interference for control of the lone star tick, Amblyomma americanum.</a> Vaccine. 28, 1786-1795 (2010).</li><li>Zivkovic, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+genes+in+salivary+glands+of+male+Rhipicephalus+(Boophilus)+microplus+in+response+to+infection+with+Anaplasma+marginale.">Differential expression of genes in salivary glands of male Rhipicephalus (Boophilus) microplus in response to infection with Anaplasma marginale.</a> BMC Genomics. 11, 186-186 (2010).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+tick+infections+with+Anaplasma+marginale+and+A.+phagocytophilum+by+targeting+the+tick+protective+antigen+subolesin.">Reduction of tick infections with Anaplasma marginale and A. phagocytophilum by targeting the tick protective antigen subolesin.</a> Parasitol. Res. 100, 85-91 (2006).</li><li>Narasimhan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+Ixodes+scapularis+anticoagulation+by+using+RNA+interference.">Disruption of Ixodes scapularis anticoagulation by using RNA interference.</a> Proc. Natl. Acad. Sci U.S.A. 101, 1141-1146 (2004).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+screening+in+ticks+for+identification+of+protective+antigens.">RNA interference screening in ticks for identification of protective antigens.</a> Parasitol. Res. 96, 137-141 (2005).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tick+protective+antigen,+4D8,+is+a+conserved+protein+involved+in+modulation+of+tick+blood+ingestion+and+reproduction.">The tick protective antigen, 4D8, is a conserved protein involved in modulation of tick blood ingestion and reproduction.</a> Vaccine. 24, 4082-4095 (2006).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+effect+of+silencing+the+expression+of+tick+protective+antigens+4D8+and+Rs86+in+Rhipicephalus+sanguineus+by+RNA+interference.">Synergistic effect of silencing the expression of tick protective antigens 4D8 and Rs86 in Rhipicephalus sanguineus by RNA interference.</a> Parasitol. Res. 99, 108-113 (2006).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocidal+control+of+ticks+by+silencing+of+a+single+gene+by+RNA+interference.">Autocidal control of ticks by silencing of a single gene by RNA interference.</a> Biochem. Biophys. Res. Commun. 344, 332-338 (2006).</li><li>Nijhof, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bm86,+Bm91+and+subolesin,+in+the+silencing+of+the+tick+protective+antigens.">Bm86, Bm91 and subolesin, in the silencing of the tick protective antigens.</a> Int. J. Parasitol. 37, 653-662 (2007).</li><li>Kocan, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+the+defensin,+varisin,+in+male+Dermacentor+variabilis+by+RNA+interference+results+in+reduced+Anaplasma+marginale+infections.">Silencing of the defensin, varisin, in male Dermacentor variabilis by RNA interference results in reduced Anaplasma marginale infections.</a> Exp. Appl. Acarol. 46, 17-28 (2008).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+the+role+of+tick+subolesin+in+gene+expression.">Evidence of the role of tick subolesin in gene expression.</a> BMC Genomics. 9, 372-372 (2008).</li><li>Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+RNA+interference+of+the+subolesin+and+voraxin+genes+in+male+Amblyomma+hebraeum+(Acari:+Ixodidae)+on+female+engorgement+and+oviposition.">The impact of RNA interference of the subolesin and voraxin genes in male Amblyomma hebraeum (Acari: Ixodidae) on female engorgement and oviposition.</a> Exp. Appl. Acarol. 47, 71-86 (2009).</li><li>Galindo, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tick+subolesin+is+an+ortholog+of+the+akirins+described+in+insects+and+vertebrates.">Tick subolesin is an ortholog of the akirins described in insects and vertebrates.</a> Dev. Comp. Immunol. 33, 612-617 (2009).</li><li>Canales, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+and+immunogenicity+of+the+mosquito+ortholog+of+the+tick+protective+antigen,+subolesin.">Conservation and immunogenicity of the mosquito ortholog of the tick protective antigen, subolesin.</a> Parasitol. Res. 105, 97-111 (2009).</li><li>Kocan, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silencing+of+genes+involved+in+Anaplasma+marginale-tick+interactions+affects+the+pathogen+developmental+cycle+in+Dermacentor+variabilis.">Silencing of genes involved in Anaplasma marginale-tick interactions affects the pathogen developmental cycle in Dermacentor variabilis.</a> BMC Dev. Biol. 9, 42-42 (2009).</li><li>Zivkovic, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subolesin+expression+in+response+to+pathogen+infection+in+ticks.">Subolesin expression in response to pathogen infection in ticks.</a> BMC Immunol. 11, 7-7 (2010).</li><li>Karim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomics+tool:+gene+silencing+in+Ixodes+scapularis+eggs+and+nymphs+by+electroporated+dsRNA.">Functional genomics tool: gene silencing in Ixodes scapularis eggs and nymphs by electroporated dsRNA.</a> BMC Biotechnol. 10, 1-1 (2010).</li><li>Kocan, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transovarial+silencing+of+the+subolesin+gene+in+three-host+ixodid+tick+species+after+injection+of+replete+females+with+subolesin+dsRNA.">Transovarial silencing of the subolesin gene in three-host ixodid tick species after injection of replete females with subolesin dsRNA.</a> Parasitol. Res. 100, 1411-1415 (2007).</li><li>de la Fuente, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+tick-pathogen+interface+for+novel+control+strategies.">Targeting the tick-pathogen interface for novel control strategies.</a> Front. Biosci. 13, 6947-6956 (2008).</li><li>Kurscheid, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+a+tick+RNAi+pathway+by+comparative+genomics+and+reverse+genetics+screen+of+targets+with+known+loss-of-function+phenotypes+in+Drosophila.">Evidence of a tick RNAi pathway by comparative genomics and reverse genetics screen of targets with known loss-of-function phenotypes in Drosophila.</a> BMC Mol. Biol. 10, 26-26 (2009).</li></ol>

利害の衝突は宣言されません。

他の方法がダニ14 33にRNAiに記載されているが、dsRNAの注射は、ここで説明する最も広く食物を与えられていない（表1）と供給チック16,25,34の両方で使用されています。 RNAiは、ダニの遺伝子機能の研究、ダニ媒介病原体のインタフェースの特性評価とスクリーニングとダニ防御抗原14,35の特性評価のための有用なツールであることが示されている。特に、RNAiは、チック35の機能解析のための最も有効なツールとなっています。 方法論的に、RNAiは、おそらく多数の個人の遺伝子のノックダウンを可能にすることがより効率的な方法に進化していきます。ダニでdsRNA誘発性RNAiのメカニズムは、この種の35,36でこの遺伝学的ア ​​プローチの理解と活用に貢献する洗練されるべきである。マダニにおけるRNAiのオフターゲット効果の程度は、完全に14,27を対処する必要がある重要な問題です。最後に、RNAiはほとんどの可能性ダニの遺伝子制御とシステム生物学の研究に総合的な貢献を提供し、ダニ - 病原体のインタフェースとダニの蔓延とダニ媒介性病原体の伝播を制御するためのワクチン開発に影響を及ぼす可能性があります。

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Access RT-PCR system</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>A1250</td>
<td>&nbsp;</td>
<tr>
<td>Purelink PCR purification kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>K3100-02</td>
<td>&nbsp;</td>
<tr>
<td>Megascript RNAi kit</td>
<td>&nbsp;</td>
<td>Ambion</td>
<td>AM1626M</td>
<td>&nbsp;</td>
<tr>
<td>Red dental wax</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>72674</td>
<td>&nbsp;</td>
<tr>
<td>Plastic cups, 1.25 oz and lids</td>
<td>&nbsp;</td>
<td>Solo Cup Company, Urbana Ill.</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fine forceps</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>Various</td>
<td>&nbsp;</td>
<tr>
<td>Insulin syringe</td>
<td>&nbsp;</td>
<td>Monoject</td>
<td>&nbsp;</td>
<td>Fitted with a &frac12;&#x201D;, 29 gauge needle</td>
</tr>
<tr>
<td>Hamilton syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>701SN,33/.375&#x201D;/45DGR</td>
<td>Custom made</td>
</tr>
<tr>
<td>TriReagent</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>93289</td>
<td>&nbsp;</td>
<tr>
<td>iScript One-Step RT-PCR Kit with SYBR Green</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>170-8892</td>
<td>&nbsp;</td>
<tr>
<td>Real-time PCR detection system</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>Several</td>
<td>Please refer to <a href="http://www.bio-rad.com/">http://www.bio-rad.com/</a></td>
</tr>		</tbody>
		</table>
		</div>

rna interference in ticks

ダニは、野生と家畜と人間の真正の吸血性外部寄生虫であり、ヒトの疾患1、畜牛業界世界中で2を影響を与える最も重要なベクトルのベクトルとしての蚊への2番目の世界的であると考えられている。チックはサブクラスダニ目、注文Parasitiformes、亜Ixodidaに分類され、北極から熱帯地域3に世界的に分布している。ダニの蔓延を制御するための努力にもかかわらず、これらの外部寄生虫は、人間と動物の健康に深刻な問題が4,5のまま。 RNA干渉（RNAi）6遺伝子機能や代謝経路への影響を判断するために、遺伝子発現の混乱を含む核酸系逆遺伝学的ア ​​プローチです。低分子干渉RNA（siRNA）は二本鎖RNA（dsRNA）とdsRNAのトリガ7月9日と同じ配列を含む細胞質mRNAの強力な配列特異的分解の結果によって開始されるRNAi経路のエフェクター分子である。 dsRNAをによって開始された転写後遺伝子サイレンシングのメカニズムがすべての真核生物で発見されたことはこれまで研究し、RNAiは、急速に機能ゲノミクス研究やその他のアプリケーション10のツールとしてさまざまな生物で開発されています。 RNAiは、遺伝子操作のための代替アプローチが利用できなかったり、信頼できない5,11であるダニや他の生物の中で最も広く使用されている遺伝子サイレンシング技術となっている。ダニの遺伝的特性は、RNAiの12,13の最近の応用まで限定されている。 RNAiは利用可能であることを短い時間で、それはダニの遺伝子機能を研究するための貴重なツール、ダニ媒介病原体のインタフェースの特性評価とスクリーニングとダニ防御抗原14の特性であることが証明されました。ここで、食物を与えられていないダニへのdsRNAの注入によるRNAiの方法が記載されている。それはこの実験的なアプローチから得られた知識は、基本的な生物学的システムの理解とダニの蔓延を制御し、ダニ媒介性病原体15-19伝送を予防するワクチンの開発に著しく寄与する可能性があります。

Watch this Scientific Journal Video about RNA Interference in Ticks at JoVE.com

RNA Interference in Ticks

食物を与えられていないダニへのdsRNAの注射により、RNA干渉（RNAi）のための方法が記載されている。 RNAiは、遺伝子操作の他のメソッドの使用が制限されているダニの中で最も広く使用されている遺伝子サイレンシング技術です。

ダニのRNA干渉

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

Research

JoVE Journal

Immunology and Infection

18.0K ビュー.  Oklahoma State University. 食物を与えられていないダニへのdsRNAの注射により、RNA干渉（RNAi）のための方法が記載されている。 RNAiは、遺伝子操作の他のメソッドの使用が制限されているダニの中で最も広く使用されている遺伝子サイレンシング技術です。

ビデオ: RNA Interference in Ticks

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

siRNA導入のための細胞型に特異的な抗HIV gp120のアプタマーの開発

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

免疫・感染学

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

マダニScapularisダニの唾液、唾液腺、および体液コレクション

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

ライム病研究における送信および外因診断法のために動物にダニの摂食

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

Using RNA-interference to Investigate the Innate Immune Response in Mouse Macrophages

Macrophages are key phagocytic innate immune cells. When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the pathogen, produce various cytokines and chemokines to recruit and stimulate other immune cells, and present antigens to stimulate the adaptive immune response. Thus, being able to efficiently manipulate macrophages with techniques such as RNA-interference (RNAi) is critical to our ability to investigate this important innate immune cell. However, macrophages can be technically challenging to transfect and can exhibit inefficient RNAi-induced gene knockdown. In this protocol, we describe methods to efficiently transfect two mouse macrophage cell lines (RAW264.7 and J774A.1) with siRNA using the Amaxa Nucleofector 96-well Shuttle System and describe procedures to maximize the effect of siRNA on gene knockdown. Moreover, the described methods are adapted to work in 96-well format, allowing for medium and high-throughput studies. To demonstrate the utility of this approach, we describe experiments that utilize RNAi to inhibit genes that regulate lipopolysaccharide (LPS)-induced cytokine production.

In this protocol, we describe methods to efficiently transfect murine macrophage cell lines with siRNAs using the Amaxa Nucleofector 96-well Shuttle System, stimulate the macrophages with lipopolysaccharide, and monitor the effects on inflammatory cytokine production.

マウスマクロファージにおける先天性免疫応答を調査するために、RNA干渉を使用

Macrophages are key phagocytic innate immune cells.  When macrophages encounter a pathogen, they produce antimicrobial proteins and compounds to kill the ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

ヌクレオカプシドアニーリング媒介電気泳動（NAME）アッセイは、HIV-1ヌクレオカプシド阻害剤の迅速な同定を可能にする

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

誘導発現系を適用すると、細胞内シグナル伝達と細菌の病原性因子の干渉を研究するために、

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. However, isolating bacterial RNA from infected cells or tissues is a challenging task, since mammalian RNA mostly dominates the lysates of infected cells. Here we describe an optimized method for RNA isolation of Listeria monocytogenes bacteria growing within bone marrow derived macrophage cells. Upon infection, cells are mildly lysed and rapidly filtered to discard most of the host proteins and RNA, while retaining intact bacteria. Next, bacterial RNA is isolated using hot phenol-SDS extraction followed by DNase treatment. The extracted RNA is suitable for gene transcription analysis by multiple techniques. This method is successfully employed in our studies of Listeria monocytogenes gene regulation during infection of macrophage cells 1-4. The protocol can be easily modified to study other bacterial pathogens and cell types.

RNA Purification from Intracellularly Grown Listeria monocytogenes in Macrophage Cells

Here we describe a method for bacterial RNA isolation from Listeria monocytogenes bacteria growing inside murine macrophages. This technique can be used with other intracellular pathogens and mammalian host cells.

RNA精製は、細胞内から成長します<em&gt;リステリア菌</em&gt;マクロファージ細胞で

Analysis of the transcriptome of bacterial pathogens during mammalian infection is a valuable tool for studying genes and factors that mediate infection. ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

次世代エレクトロポレーションによって初代ヒトTh17細胞の小さなRNAのトランスフェクション

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

全血豚で表されるコーディングおよび非コーディングの RNA クラスの識別

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

二本鎖 RNA と否定的な感覚の RNA のウイルス感染におけるパターン認識受容体の共焦点レーザー顕微鏡

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...