This research was possible through funding from the University of Neuch&#226;tel. We would like to thank all the students that helped in improving this technique, namely our colleague Kevin Thievent. We would also like to thank the members of the Thomas Lab for making their laboratory available. We would like to thank Janet Teeple for her help with mosquito rearing. We would also like to thanks Loyal Hall in the laboratory of Pr. Tom Baker for his help in the preparation of the micro capillary glass tubes.

注射や解剖1.生理食塩水<ol><li> pH値= 6.8で1.3のNaCl、0.5mMのKClと0.2 mMのCaCl 2をを得るために、蒸留水に塩化ナトリウム、塩化カリウム、および塩化カルシウムを添加することにより、生理食塩水を準備します。 </li><li>透明ビーズを着色するために生理食塩水の99ミリリットルに0.1％メチルグリーン溶液1mlを追加します。これは、0.001％メチルグリーン」注射液」です。 </li><li>その後、生理食塩水45mlの0.1％メチルグリーン溶液5mlを追加します。この0.01％メチルグリーン」解剖溶液」切開中のビーズの着色とその観察を容易にするために、メチルグリーンで10倍に濃縮します。 注：必要に応じてバッファを殺菌フィルター。 </li></ol> 2.キャピラリーの準備<ol><li>熱プルマイクロキャピラリーガラス管最小のビーズ（φ= 40μm）の42よりもわずかに大きい非常に微細な先端を得ることができます。 </li><li>オープンとAピンセットでチップを破壊したり、先端をオフにソーイングすることにより、各キャピラリをdjust。 </li></ol> 3.蚊飼育<ol><li> 26±1℃でリアのA.ハマダラカ 、70±5％の相対湿度、12：12時間の明：暗サイクル。成人女性を維持し、それらを10％糖液へのアクセスを与えます。 </li></ol>キャピラリー4.ビーズ選択<ol><li> 5センチメートルペトリ皿に負に帯電したビーズ（直径40〜120ミクロン）の0.009グラムを注ぎ、事前に注射液の30分の5ミリリットルを追加します。 注：目標は、注射のためのビーズ選択を容易にするために、染料の色にビーズをできるようにすることです。彼らは無菌ジャーに来ていない場合は、ビーズをオートクレーブ。 </li><li>キャピラリーピペット電球に搭載されたキャピラリーを用いて、視覚的に双眼顕微鏡下での生理食塩水0.1μlの最小のビーズを選択します。 注：同時に複数のビーズを接種すると、0.1マイクロリットルの総容積から3ビーズを選択します。</li></ol> 5.モスキート処理および接種<ol><li>昆虫吸引器を使用して、50ミリリットルチューブに各蚊を置きます。 </li><li>砕いた氷（2-5分）にチューブを配置することによって、簡単に各蚊を冷やします。 </li><li>双眼顕微鏡下でその右側に蚊を置きます。 </li><li>実体顕微鏡下で、しっかりと胸腔のキューティクルの左側を貫通した後、液体やビーズを注入することにより、キャピラリーで蚊を接種します。 注：可能な限り蚊に同様に垂直にキャピラリーを保持することにより、飛翔筋を傷つけないように注意してください。 </li><li>毛細血管を削除します。 </li><li>フェザー級昆虫学の鉗子を使用して、蚊帳で覆われた180ミリリットルのプラスチックカップに個別に女性を配置。彼らに10％の糖溶液へのアクセスを与えます。 注：蚊は、糖液滴にはまり込むことができるように注意、糖液で必要とされます。 </li></ol> 6.モスキート解剖とビーズの写真<ol><li>蚊が接種後24時間生きているかどうかを確認してください。もしそうであれば、その状態を確認し、飛ぶことができますだけで蚊を保ちます。 </li><li> -20℃で、これらの蚊をフリーズします。 注：必要に応じて彼らは、解剖の前に数ヶ月に数日間維持することができます。 </li><li> 32Xの倍率で双眼顕微鏡下でピンセットを使って蚊羽を削除します。透明テープでスライドガラス上に翼を固定してください。各個々の蚊のために使用されるコードを書き留めます。 </li><li>スキャナでガラススライドを入れて、1200 dpiでそれらをスキャンします。 注：コードは、個々の蚊のために読み取り可能であることを確認してください。 </li><li>ガラス顕微鏡スライド上にと解剖溶液中で、頭部と腹部から胸部を分離するために鉗子を使用しています。 </li><li>ビーズを取得するために鉗子で胸部を開きます。 注：腹部に移動しないビーズは、互いに接触しておらず、それらのほとんどがTで自由に浮きます彼は血リンパ。非メラニンビーズを見つけるのは厳しいものになる可能性があるため、ピンセットでビーズを壊さないように注意してください。色を得るためにビーズ1〜5分間待ってください。 </li><li>写真を撮る前に、ガラス顕微鏡スライド上の解剖液の液滴中にビーズを転送します。 注：ビーズを洗浄する必要はありません。 </li><li>カメラを備えた顕微鏡を使用して、標準の照明設定と400Xの倍率で各ビーズのデジタル画像を撮ります。 注：彼らの比較を可能にするために、異なる画像間の照明を変更しないでください。 </li></ol> 7.画像解析：ビーズメラニン<ol><li>画像解析ソフトウェアを開きます。 「ファイル」をクリックして、「開く」をビーズ画像を見つけること。 </li><li>クリックして「分析」。クリックして「設定の測定値。 &quot; 「セット測定」ウィンドウで「グレイ平均値」ボックスにチェックを入れ。 </li><li>サークル選択ツールアイコンをクリックします。ビーズ周囲を選択します。に残りますビーズを左右し、その周りに明るいハローを避けます。 </li><li> 「分析」メニューをクリックします。クリックして「尺度。」ビーズの断面積の平均グレー値とサイズを取得します。 <ol><li> 「 - 灰色の平均値256。」：各ビーズのメラニン値を取得するには、電卓や計算機のソフトウェアを使用して、次の操作を行いますメラニンの尺度は0が完全に白画像を表すと、0と256の間の値です。 </li></ol></li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Starvation+Reveals+Maintenance+Cost+of+Humoral+Immunity.">Starvation Reveals Maintenance Cost of Humoral Immunity.</a> Evol Biol. 37 (1), 49-57 (2010).</li><li>Sheldon, B. C., Verhulst, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological+immunology:+costly+parasite+defences+and+trade-offs+in+evolutionary+ecology.">Ecological immunology: costly parasite defences and trade-offs in evolutionary ecology.</a> Trend Ecol Evo. 11 (8), 317-321 (1996).</li><li>Graham, A. L., Allen, J. E., Read, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+causes+and+consequences+of+immunopathology.">Evolutionary causes and consequences of immunopathology.</a> Annu Rev Ecol Evol S. 36, 373-397 (2005).</li><li>Best, A., Long, G., White, A., Boots, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+implications+of+immunopathology+for+parasite+evolution.">The implications of immunopathology for parasite evolution.</a> P Roy Soc B-Biol Sci. 279 (1741), 3234-3240 (2012).</li><li>Cator, L. J., George, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=34;Manipulation&amp;#34;+without+the+parasite:+altered+feeding+behaviour+of+mosquitoes+is+not+dependent+on+infection+with+malaria+parasites.">34;Manipulation&amp;#34; without the parasite: altered feeding behaviour of mosquitoes is not dependent on infection with malaria parasites.</a> P Roy Soc B-Biol Sci. 280 (1763), 20130711 (2013).</li><li>Voordouw, M. J., Lambrechts, L., Koella, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+maternal+effects+after+stimulation+of+the+melanization+response+in+the+yellow+fever+mosquito+Aedes+aegypti.">No maternal effects after stimulation of the melanization response in the yellow fever mosquito Aedes aegypti.</a> Oikos. 117 (8), 1269-1279 (2008).</li><li>Paskewitz, S., Riehle, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+Plasmodium+refractory+and+susceptible+strains+of+Anopheles+gambiae+to+inoculated+Sephadex+beads.">Response of Plasmodium refractory and susceptible strains of Anopheles gambiae to inoculated Sephadex beads.</a> Dev comp immunol. 18 (5), 369-375 (1994).</li></ol>

The authors declare that they have no competing financial interests.

この注入法は、蚊におけるメラニンの応答を刺激し、検討することが有用です。例えば、ここでは、免疫刺激負荷の影響を検討しました。 この手順の重要なステップは、適切に蚊に接種することです。飛翔筋または蚊自体への過度な損傷が給紙から蚊を防ぐことができるか、解剖の前にそれを殺すことができます。第二の重要なステップは、それらを殺すことなく、そ?...

そのキューティクルまたはそれらの中腸上皮4を通して破る3 -昆虫は寄生虫や病原体1から身を守るために免疫応答に依存しています。蚊では、これらの応答は、細菌5、ウイルス6、フィラリア線虫7、およびマラリア原虫1,8,9に対して効率的です。 12 -蚊では、キー免疫応答は、メラニン10で異物をカプセル化したものです。 12 -このカプセル化は、中腸内または血リンパ循環システム10に発生する可能性があります。このメラニン応答は、プロフェノールオキシダーゼカスケード10の結果である- 12、そしてそれは寄生虫の死に、またはそれらの食作用につながることができます。血球細胞の数は限られている大人の蚊では、メラニンは、マラリア原虫の寄生虫やフィラリア線虫7に対してのように、体液性応答です。いくつかの他の昆虫では、それは直接7メラニン化するために寄生虫の周りに集まる血球細胞です。また、メラニンは7治癒産卵とキューティクルの傷のようないくつかの他の生理学的プロセスのためにも不可欠です。 18 -免疫応答の刺激は、いくつかの農業および公衆衛生モデル系13の昆虫耐性を研究するためのツールとして使用されます。 16,19 -宿主-寄生虫相互作用14を研究するハマダラカ蚊、アフリカにおけるマラリアの主要なベクトルで使用されています。これらの技術は、それらのパターン認識受容体（PRR）2で寄生虫を検出するために、昆虫の能力に基づいています。蚊はまた、病原体関連分子パターン（PAMP）のようなそれらの生物学的に干渉する他の分子を検出する、またはコラーゲンおよび核酸の放出に起因する独自の損傷した細胞を検出することができます。蚊免疫細胞23 -このような血球などの検出20のために使用されています。主な免疫シグナル伝達経路は、IMD、トール、JAK / STAT 24、およびリボ核酸干渉（RNAi）25,26です。トールおよびIMD経路の両方は、メラニンの応答に影響を及ぼし、プロフェノールオキシダーゼカスケード10との対話- 12。 メラニンの応答を刺激するために使用される標準的なツールは、胸腔の血リンパ中に小さなビーズと蚊の接種です。メラニンのカプセル化の程度は、その後、蚊の切開を介してビーズを回収した後、19を測定することができます。ほとんどの研究では、唯一のビーズは蚊15,16,27毎に注入したが、より多くのビーズを注入すると、メラニン応答19の限界を研究するために可能です。これらのビーズは、蚊の生理機能の妨害を制限するために、注射液（生理血清）を使用して注入され、蚊15,16,27の乾燥。色素は、ビーズの選択を容易にするためにこの溶液に添加します。これは、ビーズ15,16,27を取得するために使用される解剖ソリューションについても同様です。 非病原性の刺激で虫を接種することの利点は、免疫応答に直接影響に焦点を当てることができることです。 31、または免疫回避31 - - 34による寄生病原28、免疫抑制29への複雑な効果はありません。また、このような寿命や産卵数などの他の生活史形質、上の刺激の結果は、また、研究することができます。したがって、進化生態学を研究する研究者は、このようなツール2,35,36が必要な場合があります。例えば、免疫挑戦マルハナバチは、飢餓の下に短縮寿命を持っています。免疫刺激と展開の同様の負の効果は、多くの場合、短いで、その結果、別の無脊椎動物のモデルで観察されていますえー寿命以下繁殖成功13,27,37。このような研究は、環境に2,4,38を変えて実施することができます。免疫力を刺激することも免疫病理39,40に直接焦点を当てたものに興味があります。 このプロトコルは、メラニン応答を刺激し、直接メラニンの量を測定するために蚊を有するビーズの接種に基づいています。これは、異なる実験設定でメラニン応答の定量的および定性的な研究を可能にします。このようなツールは、加熱死菌41に抗菌性応答などの他の免疫応答の刺激に拡張することができます。また、多くの生態系の設定で行うことができます。

<table><tbody><tr><td>Microcapillary glass tubes GB120TF-10</td><td>science-products.com</td><td>GB120TF-10</td><td>http://www.science-products.com/Products/CatalogG/Glass/Glass.html</td></tr><tr><td>Microcaps Capillary pipette bulb</td><td>Drumond</td><td>1-000-9000</td><td></td></tr><tr><td>negatively charged Sephadex CM C-25 beads</td><td>Sigma-Aldrich, Steinheim, Germany</td><td>C25120 SIGMA</td><td>need few to start</td></tr><tr><td>Methyl green</td><td>Sigma-Aldrich</td><td>323829 ALDRICH</td><td>need few to start</td></tr><tr><td>Software ImageJ</td><td>opensource</td><td></td><td>Version 1.47f7 or later</td></tr></tbody></table>

inoculating anopheles gambiae mosquitoes with beads to induce and measure the melanization immune response

免疫応答の刺激は、有効性と免疫のメカニズムを検討する無脊椎動物研究における一般的なツールです。粒子は免疫系によって検出され、免疫エフェクターの産生を誘導するように、この刺激は、昆虫への非病原性粒子の注射に基づいています。私たちは、蚊ハマダラカにおけるメラニンの応答の刺激にここに焦点を当てます。メラニンの応答は、メラニンの暗い層で異物や寄生虫のカプセル化につながります。この応答を刺激するために、蚊は、微小ガラス管を用いて胸腔内のビーズを用いて接種します。そして、24時間後に、蚊がビーズを取得するために解剖されています。ビーズのメラニンの程度は画像分析ソフトウエアを用いて測定されます。ビーズは、病原性寄生虫の効果、または免疫応答を回避または抑制するための能力を持っていません。これらの注射は、measuする方法です免疫有効性及びそのような繁殖力や長寿などの他の生活史形質、上の免疫刺激の影響再。これは、直接宿主 - 寄生虫相互作用を研究するとまったく同じではありませんが、免疫力とその進化生態学を研究するための興味深いツールです。

<html>いくつかのビーズが少ない他よりもメラニン（ 図1）で覆われていたとして蚊は、同じようにビーズをメラニン化するすべてではないでした。実際、いくつかのビーズが（ 図1）他の人が完全に暗くしたのに対し、理由メラニンの不足の青のままでした。メラニン値（青およびunmelanizedビーズに相当）が0と100（暗いと高メラニンビーズに?...

Watch this Scientific Journal Video about Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response at JoVE.com

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

接種<em&gt;ハマダラカ</emメラニン免疫応答を誘導し、測定するためにビーズと&gt;蚊

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is ...

inoculating-anopheles-gambiae-mosquitoes-with-beads-to-induce-measure

Research

JoVE Journal

Immunology and Infection

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

7.9K ビュー.  Université de Neuchâtel. Through inoculation with beads, the described technique enables the stimulation of the mosquito melanization response in the hemolymph circulating system. The amount of melanin covering the beads can be measured after dissection as a measure of the immune response.

ビデオ: Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Injection of An. stephensi Embryos to Generate Malaria-resistant Mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.

Anopheles stephensi mosquitoes are vectors for malaria inhabiting India and throughout Asia. This video demonstrates the technique for performing microinjections of this species with transgenes that will confer resistance to the malaria to the mosquito. Much of the methodology demonstrated in this video is applicable to microinjection techniques of other mosquito species.

の注入。ハマダラカの胚は、マラリア耐性の蚊を生成する

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This ...

生物学

Site-Directed &#966;C31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the genome of Anopheles mosquitoes. Amongst these methods, the &#966;C31 system allows precise and stable site-directed integration of transgenes, or the substitution of integrated transgenic cassettes via recombinase-mediated cassette exchange (RMCE). This method relies on the action of the Streptomyces &#966;C31 bacteriophage integrase to catalyze recombination between two specific attachment sites designated attP (derived from the phage) and attB (derived from the host bacterium). The system uses one or two attP sites that have been integrated previously into the mosquito genome and attB site(s) in the donor template DNA. Here we illustrate how to stably modify the genome of attP-bearing Anopheles docking lines using two plasmids: an attB-tagged donor carrying the integration or exchange template and a helper plasmid encoding the &#966;C31 integrase. We report two representative results of &#966;C31-mediated site-directed modification: the single integration of a transgenic cassette in An. stephensi and RMCE in An. gambiae mosquitoes. &#966;C31-mediated genome manipulation offers the advantage of reproducible transgene expression from validated, fitness neutral genomic sites, allowing comparative qualitative and quantitative analyses of phenotypes. The site-directed nature of the integration also substantially simplifies the validation of the single insertion site and the mating scheme to obtain a stable transgenic line. These and other characteristics make the &#966;C31 system an essential component of the genetic toolkit for the transgenic manipulation of malaria mosquitoes and other insect vectors.

The protocol describes how to achieve site-directed modifications in the genome of Anopheles malaria mosquitoes using the &#966;C31 system. Modifications described include both the integration and the exchange of transgenic cassettes in the genome of attP-bearing docking lines.

マラリアのアノフェレスベクターにおける部位特異的φC31媒介集積分とカセット交換

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the ...

遺伝学

Microinjection Method for Anopheles gambiae Embryos

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA fragments encoding genes of interest as well as favorable traits into the insect germline in a stable and heritable manner. The resulting transgenic strains can be studied for phenotypic changes resulting from the expression of the integrated DNA to answer basic questions or used in practical applications. Although the technology is straightforward, it requires of the investigator patience and practice to achieve a level of skill that maximizes efficiency. Shown here is a method for microinjection of embryos of the African malaria mosquito, Anopheles gambiae. The objective is to deliver by microinjection exogenous DNA to the embryo so that it can be taken up in the developing germline (pole) cells. Expression from the injected DNA of transposases, integrases, recombinases, or other nucleases (for example CRISPR-associated proteins, Cas) can trigger events that lead to its covalent insertion into chromosomes. Transgenic An. gambiae generated from these technologies have been used for basic studies of immune system components, genes involved in blood-feeding, and elements of the olfactory system. In addition, these techniques have been used to produce An. gambiae strains with traits that may help control the transmission of malaria parasites.

Microinjection Method for Anopheles gambiae Embryos

Microinjection techniques are essential to introduce exogenous genes into the genomes of mosquitoes. This protocol explains a method used by the James laboratory to microinject DNA constructs into Anopheles gambiae embryos to generate transformed mosquitoes.

アノフェレスガンビア胚の微小注入法

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA ...

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood. 
We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility. 
All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

アン<em&gt;インビトロ</em&gt; HIV重感染のコンテキストでのマラリアに対する免疫応答を測定するためのモデル

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

免疫・感染学

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

弱毒で皮内免疫後のマウスの皮膚及び排出リンパノードから骨髄性細胞の単離<em&gt;マラリア原虫</em&gt;スポロゾイト

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

磁気共鳴映像法を用いた実験的大脳マラリアモデルにおける浮腫発生と微小血管病変のin vivo追跡

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

医学

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This parasitic disease requires a protozoan parasite, an Anopheles mosquito vector, and a vertebrate host. The Anopheles genus comprises more than 500 species, of which 60 are known as vectors of the parasite. The Plasmodium parasite genus consists of 250 species, and 48 of these are involved in disease transmission. Furthermore, the Plasmodium falciparum parasite has contributed toward an estimated 99.7% of malaria cases in sub-Saharan Africa in recent years. 
Gametocytes form part of the sexual stage of the parasite and are ingested by the female mosquito upon feeding on an infected human host. Further development of the parasite within the mosquito is enhanced by favorable environmental conditions in the midgut of the mosquito. Here, the fusion of the female and male gametes takes place, and the motile ookinetes originate. The ookinetes enter the midgut epithelium of the mosquito, and mature ookinetes form oocysts, which, in turn, produce motile sporozoites. These sporozoites migrate to the mosquito's salivary glands and are injected as a mosquito takes a blood meal. 
For drug discovery purposes, mosquitoes were artificially infected with gametocyte-infected blood in the standard membrane feeding assay (SMFA). To detect infection within the mosquito and/or to assess the efficacy of antimalarial compounds, the midguts of the female mosquitoes were removed post infection and were stained with mercurochrome. This method was used to enhance the visual detection of oocysts under the microscope for the accurate determination of oocyst prevalence and intensity.

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

The standard membrane feeding assay (SMFA) is regarded as the gold standard for the assessment and identification of potential antimalarial compounds. This artificial feeding system is used to infect mosquitoes to further evaluate the effects of such compounds on the intensity and prevalence of the Plasmodium falciparum parasite.

ハマダラカ媒介動物における熱帯熱マラリア原虫感染の検出のための標準的な膜摂食アッセイ

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This ...

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is transmitted through inoculation of the sporozoite stage of Plasmodium by infected mosquitoes. Transgenic Plasmodium has allowed us to understand the biology of malaria better and has contributed directly to malaria vaccine development efforts. Here, we describe a streamlined methodology to generate transgenic Plasmodium berghei sporozoites.

遺伝子組換え マラリア原虫ベルヘイ ・スポロゾイトの生成

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of ...

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory for functional genomic studies. The ability to knockdown individual genes selectively via this reverse genetic technique has allowed many researchers to rapidly uncover the biological roles of numerous genes within many organisms, by evaluation of loss-of-function phenotypes. In the major human malaria vector Anopheles gambiae, the predominant method used to reduce the function of targeted genes involves injection of double-stranded (dsRNA) into the hemocoel of the adult mosquito. While this method has been successful, gene knockdown in adults excludes the functional assessment of genes that are expressed and potentially play roles during pre-adult stages, as well as genes that are expressed in limited numbers of cells in adult mosquitoes. We describe a method for the injection of Serine Protease Inhibitor 2 (SRPN2) dsRNA during the early pupal stage and validate SRPN2 protein knockdown by observing decreased target protein levels and the formation of melanotic pseudo-tumors in SRPN2 knockdown adult mosquitoes. This evident phenotype has been described previously for adult stage knockdown of SRPN2 function, and we have recapitulated this adult phenotype by SRPN2 knockdown initiated during pupal development. When used in conjunction with a dye-labeled dsRNA solution, this technique enables easy visualization by simple light microscopy of injection quality and distribution of dsRNA in the hemocoel.

RNAi Trigger Delivery into Anopheles gambiae Pupae

RNA interference (RNAi) is an extremely valuable tool for uncovering gene function. However, the ability to target genes using RNAi during pre-adult stages is limited in the major human malaria vector Anopheles gambiae. We describe an RNAi protocol to reduce gene function via direct injection during pupal development.

RNAiのトリガ配信へ<em&gt;ハマダラカ</em&gt;蛹

RNA interference (RNAi), a naturally occurring phenomenon in eukaryotic organisms, is an extremely valuable tool that can be utilized in the laboratory ...

Effective Oral RNA Interference (RNAi) Administration to Adult Anopheles gambiae Mosquitoes

RNA interference has been a heavily utilized tool for reverse genetic analysis for two decades. In adult mosquitoes, double-stranded RNA (dsRNA) administration has been accomplished primarily via injection, which requires significant time and is not suitable for field applications. To overcome these limitations, here we present a more efficient method for robust activation of RNAi by oral delivery of dsRNA to adult Anopheles gambiae. Long dsRNAs were produced in Escherichia coli strain HT115 (DE3), and a concentrated suspension of heat-killed dsRNA-containing bacteria in 10% sucrose was offered on cotton balls ad-libitum to adult mosquitoes. Cotton balls were replaced every 2 days for the duration of the treatment. Use of this method to target doublesex (a gene involved in sex differentiation) or fork head (which encodes a salivary gland transcription factor) resulted in reduced target gene expression and/or protein immunofluorescence signal, as measured by quantitative Real-Time PCR (qRT-PCR) or fluorescence confocal microscopy, respectively. Defects in salivary gland morphology were also observed. This highly flexible, user-friendly, low-cost, time-efficient method of dsRNA delivery could be broadly applicable to target genes important for insect vector physiology and beyond.

Effective Oral RNA Interference RNAi Administration to Adult Anopheles gambiae Mosquitoes

The oral administration of dsRNA produced by bacteria, a delivery method for RNA interference (RNAi) that is routinely used in Caenorhabditis elegans, was successfully applied here to adult mosquitoes. Our method allows for robust reverse genetics studies and transmission-blocking vector studies without the use of injection.

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この動画の章

の注入。ハマダラカの胚は、マラリア耐性の蚊を生成する

マラリアのアノフェレスベクターにおける部位特異的φC31媒介集積分とカセット交換

アノフェレスガンビア胚の微小注入法

アン

弱毒で皮内免疫後のマウスの皮膚及び排出リンパノードから骨髄性細胞の単離

磁気共鳴映像法を用いた実験的大脳マラリアモデルにおける浮腫発生と微小血管病変のin vivo追跡

ハマダラカ媒介動物における熱帯熱マラリア原虫感染の検出のための標準的な膜摂食アッセイ

遺伝子組換えマラリア原虫ベルヘイ・スポロゾイトの生成

RNAiのトリガ配信へ

成虫のアノフェレスガンビアエ蚊への効果的な経口RNA干渉(RNAi)投与

接種

要約

さらに動画を探す

この動画の章

の注入。ハマダラカの胚は、マラリア耐性の蚊を生成する

マラリアのアノフェレスベクターにおける部位特異的φC31媒介集積分とカセット交換

アノフェレスガンビア胚の微小注入法

アン

弱毒で皮内免疫後のマウスの皮膚及び排出リンパノードから骨髄性細胞の単離

磁気共鳴映像法を用いた実験的大脳マラリアモデルにおける浮腫発生と微小血管病変のin vivo追跡

ハマダラカ媒介動物における熱帯熱マラリア原虫感染の検出のための標準的な膜摂食アッセイ

遺伝子組換え マラリア原虫ベルヘイ ・スポロゾイトの生成

RNAiのトリガ配信へ

成虫 のアノフェレスガンビアエ 蚊への効果的な経口RNA干渉(RNAi)投与

遺伝子組換えマラリア原虫ベルヘイ・スポロゾイトの生成

成虫のアノフェレスガンビアエ蚊への効果的な経口RNA干渉(RNAi)投与