写真についてミシェル月 (ヴァンダービルト大学) に感謝したいと思います。PA と SSLB にヴァンダービルト大学提供のサポートは、地区ガバナーエレクト ・ 1445197 によってサポートされます。

<ol>
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著者が明らかに何もありません。

長いこと、ディアボルスA. neriiパターンと植物の防御、特に化学隔離18,37への抵抗のメカニズムに洞察力を提供できるが認識されています。ゲノム リソースの数は、 A. nerii10, A. neriiを使用して、モデルとして生態学的機能のゲノム研究の新しい機会を提供するため最近浮上しています。アブラムシ、植物文化のこの種の今後の作業がられるゲノムおよび機能の生態学的アプローチを活用した研究を伴う可能性が高いことを前提に、分子/ゲノム技術の基本的なプロトコルの概要を説明します。カルデノライド解毒とA. neriiで隔離の意義とメカニズムについて多くの未解決の問題が残っています。RNAi などの方法によってノックダウン式または遺伝子編集アプローチをこの点で貴重な証明します。
アブラムシを培養する際の課題の 1 つは繁殖と分散戦略のための驚異的な能力です。同質遺伝子系統が実験のために必要な場合、これらの特徴は、なぜ彼らが深刻な作物害虫のアブラムシの文化として、ほぼ毎日注意が必要ことを意味に直接関係するよくとして細心の注意です。遺伝子発現の分析のためのデータを生成するためのそれらを含む上記で説明したテクニック、アブラムシの飼育と分子解析のための一般的なプロトコルと同様に、提供十分な生物を生成する特定のステップ バイ ステップ ガイド分子および生態学的なアプリケーションの多様なセットのa. neriiの材料。
このためには、機能または生態学的なゲノム研究はA. neriiのための地平線上にある場合、完全に彼らを提供する実験の機会を活用する生活文化と結合されるこれら必要があります。草食昆虫が彼らのホスト植物複雑なコミュニティに住んでいるし、種間相互作用40と同様、両方の種内38,39形宿主植物にA. neriiの最終的な応答.A. neriiホスト植物に特化、発散の生活史戦略15、21、カップリングと純粋な genomic または生理学的アプローチの重要性を強調を表す植物の多様なセットを表すA. neriiコミュニティの自然発生する変化の実験操作。ここで説明する方法は、 A. neriiと有毒な宿主植物との相互作用に関する機能と生態学的なゲノムの観点のためのポイントを始めています。

アブラムシは、世界中の多様な植物家族の植民地 hemimetabolous、小さな昆虫です。最も特にから不足している栄養素を供給する酵母や細菌の共生とその偏栄養共生と離散 polyphenisms 循環単為生殖を含む複雑なライフ サイクルのいくつかの機能の独特であります。sap の1工場の彼らの食事療法。ほとんどアブラムシはホスト植物の専門家、いくつか種がどちらか直接にある作物で、かなりの経済ダメージを与える重要な作物害虫や病原体やウイルスを介して彼らはベクトル2。最初アブラムシにおけるゲノム学 2010 年エンドウ アブラムシエンドウヒゲナガアブラムシ3、パブリケーション アブラムシの生物学の研究で重要なマイルス トーンをマークは、昆虫のアドレス指定についての遺伝子資源を提供されているため良いコントロールの戦略4につながる可能性のあるものを含む、草食性のライフ スタイルに適応します。それ以来、追加ゲノム リソース大豆アブラムシワタアブラムシ グリシン5注釈ゲノムと別の 3 アブラムシ種 (モモアカアブラムシの公に利用可能な全ゲノム リソースのパブリケーションと蓄積してきたcerasi (ブラック チェリー アブラムシ)、モモアカアブラムシ(桃ジャガイモ アブラムシ)した padi (鳥チェリー エンバク アブラムシ)6。貴重なデ novoトランスクリプトーム リソースが他のアブラムシの数も利用可能 (e.g.,Aphis gossypii (綿アブラムシ)7, Sitobion 褐(穀物アブラムシ)8, Cinarapinitabulaeformis (松アブラムシ)9、ワタアブラムシ nerii (トウワタ キョウチクトウ アブラムシ)10)。
アブラムシは植物11植物昆虫相互作用と生命の生態学の私達の理解に永続的な貢献をしたも。アブラムシが特に重要な貢献をした 1 つの領域は、宿主植物の化学生態学の私達の理解にあります。共同植物の防御、自分の選ぶも克服する植物の防御、およびいくつかの草食性昆虫表現多様な適応は利益12,13,14です。たとえば、トウワタ キョウチクトウ モモアカアブラムシ、ワタアブラムシ neriiは、明るい黄色、侵襲的なアブラムシ世界中の温帯、熱帯地域は、トウワタ家族 (キョウチクトウ科) 植物の定着です。キョウチクトウ科の植物は、乳状のラテックスとカチオン キャリア Na, K-atpase をバインドし、ジェネラ リスト草食動物15,に効果的な抑止力は、cardenolides として知られている強心配糖体などを含む、多様な化学防御を進化しています。16. トウワタ専門家エクスプレス、cardenolides に対する抵抗性のさまざまなモード、選択的または受動的蓄積や捕食を阻止するための手段としてまたは他の利点の17のための組織の cardenolides を変更あり。A. neriiはこの方法で cardenolides を隔離するメカニズムと機能上の利点のまま不明10,18です。
手で、ゲノム リソース、 A. neriiを提供します優れた実験モデル有毒な宿主植物との専門家の化学生態学的相互作用に関与する分子および遺伝のメカニズムの調査のため草食動物。A. nerii cardenolides19, その時以来の隔離に焦点を当てたの初期の研究のいくつか、 a. neriiの研究が広範な進化と生態学的な質問に洞察力を提供している注目に値する侵襲的な昆虫20と草食動物密度21のボトムアップとトップダウンの規則間の相互作用の遺伝的構造を含みます。A. neriiはこうして特に広範な昆虫植物相互作用の研究のための実験モデルとしての良い候補です。重要なA. neriiと任意の研究の成功に注意カルチャですアブラムシの植物のアブラムシが依存の文化を含む集団として高品質弔データの効率的な生成。私たちの目標は、両方を介してリーダーを指導することです。世代やメンテナンス、温室効果と研究所、DNA と RNA の抽出、マイクロ サテライト解析、 de novoトランスクリプトーム アセンブリおよび注釈、トランスクリプトームで植物とアブラムシの文化のための方法を以下に発現解析と qPCR 性遺伝子が発現。一方、これらのメソッドは、 A. neriiの書かれている、培養、抽出、および分析の一般的な方法をさまざまな種のアブラムシに拡張できます。

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		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:64px;">2x 4&#34; Round Standard Pot</td>
			<td>Anderson Pots</td>
			<td style="width:184px;">1503</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:64px;">DreamTaq DNA Polymerase</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:184px;">EP0701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:64px;">Trizol</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:184px;">15596026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SuperScript&#174; III First-Strand Synthesis kit</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:184px;">18080051</td>
			<td></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:64px;">Power SYBR Green PCR Master Mix</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:184px;">4367659</td>
			<td></td>
		</tr>
	</tbody>
</table>

maintaining biological cultures and measuring gene expression in aphis nerii: a non-model system for plant-insect interactions

アブラムシは宿主植物と昆虫の相互作用をめぐる質問に共生の進化と polyphenisms の開発に至る生物の質問の様々 な優れた実験的モデルです。アブラムシ数種の遺伝子資源があり、非モデル生物のゲノムを欠いているに延長されるトランスクリプトーム研究次世代シーケンシングの進化により、です。さらに、アブラムシの文化はフィールドから収集し、生態学的な遺伝学の間のギャップを埋めるための生物・分子の実験で使用する実験室で飼育できます。最後に、多くのアブラムシは無性再現遺伝子型の比較を可能にする永久的な単為のライフ サイクルでの優先ホスト植物研究所で維持できます。ワタアブラムシ nerii、トウワタ キョウチクトウ アブラムシは、生物分子の実験を使用して有毒な植物と昆虫の相互作用を研究するようなの 1 つのモデルを提供します。世代と温室効果と研究所、DNA および RNA の抽出、マイクロ サテライト解析、 de novoトランスクリプトーム アセンブリおよび注釈、トランスクリプトーム発現の植物とアブラムシの文化の維持の方法分析、および特異的発現遺伝子の qPCR 検証は概説し、ここで説明します。

植物の文化:種子は季節に応じて約 2 〜 4 週間の時間がかかります、大きく十分に鉢植え (図 1A)。再鉢植え苗は、アブラムシの文化 (図 1B) の最適なサイズに成長する別の 2 〜 4 週間かかります。
アブラムシ文化:大人A. neriiいくつかの暗い馬尾によって区別され、翼のあるかもしれない (無翅、図 3A、 B) または翼 (翼、図 3C, D)。ニンフ (図 3E, F) の 3 齢に達するとき、発展途上の翼パッドが表示されます。株式文化ベスト 1 ~ 3 の中間齢と 1 つ大人高齢者 unwinged アブラムシ; 転送によって維持されます。これにより、健康年齢人口を混合します。実験に使用する集団は、(2.4) を上記のように unwinged アブラムシを使用して培養する必要があります。A. nerii大人 1 人産むことができます 3-10 日あたり、宿主植物とアブラムシ10の年齢によって異なります。
DNA および RNA の抽出:シングル、大人A. neriiを行えば、およそ 100-200 ng/μ L の DNA (80 μ L の溶出。図 4A) および 150-300 ng/μ L の RNA (30 μ L の溶出。図 4B). 代表マイクロ サテライト ピークは、図 5に示します。3 つの条件 (制御、治療 1、処理 2) 下候補遺伝子の代表的な相対式は表 2に計算され、図 6に示します。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58044/58044fig1.jpg"> 図 1: アブラムシ文化の代表的な植物。彼らは真の葉の最初の完全なセットを開発した後 (A) 苗は鉢植え再することができます。(B) 植物は、彼らは真の葉の 3-4 セットを開発しているとき、アブラムシ文化の使用できます。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58044/58044fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58044/58044fig2.jpg"> 図 2: アブラムシを培養するために使用するツールの例です。(A) 口のピペットは、3/16"x 1/4" 外径チューブ、1,000 μ L ピペット チップ 200 μ L ピペット チップ ID を使用して作成できます。(B、C)カップ ケージ (上部を切断し、細かいメッシュで保護と透明なプラスチック カップ) を使用して、アブラムシ文化用 4 インチの鍋の上に合わせて、確実に。これは十分なライトおよび換気のためアブラムシおよび植物に適した環境を作成することができます、含まれているアブラムシを続けています。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58044/58044fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58044/58044fig3.jpg"> 図 3: 代表的な大人とワタアブラムシ neriiニンフ。(A, B)(Unwinged) 大人の無翅A. neriiは、後部端に暗い馬尾によって識別されます。(C, D)翼 (翼) 大人が十分に発達した翼によって識別され、暗く、後方で馬尾。(E, F)発展途上のA. neriiニンフが 4 齢段階を経るし、開発翼パッドは 3 齢段階で明らかになります。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58044/58044fig3large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58044/58044fig4.jpg"> 図 4: 代表的なゲル。(A) DNA 抽出 (はしご 1 kb)。7 A. nerii DNA の抽出に 3-9 のレーンで視覚化されます。否定的な制御は車線 10 で。(B) RNA の抽出を行11 A. nerii RNA の抽出に 3-13 のレーンで視覚化されます。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58044/58044fig4large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58044/58044fig5.jpg"> 図 5: マイクロ サテライト代表ピーク。タグ付き FAM の 6 ピークに青で視覚化されます。リズ 500 はしごがオレンジ色で表示されます。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58044/58044fig5large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58044/58044fig6.jpg"> 図 6: 特異的発現遺伝子の qPCR 検証します。代表的な相対的な量 (RQ) 発現 (表 2ΔΔCt メソッドを使用して計算される) 3 つの条件の下で興味の候補遺伝子の表示: 制御、処理 1、処理 2。グラフでは、1 と 2 のコントロール (表 2) に比較して治療の下候補遺伝子の発現の低下を示しています。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58044/58044fig6large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>プライマー名</td>
			<td>方向</td>
			<td>シーケンス (5' 3')</td>
		</tr>
<tr>
<td>Ago24_F</td>
			<td>楽しみにして</td>
			<td>TTTTCCCGGCACACCGAGT</td>
		</tr>
<tr>
<td>Ago24_R</td>
			<td>リバース</td>
			<td>GCCAAACTTTACACCCCGC</td>
		</tr>
<tr>
<td>前 53_F</td>
			<td>楽しみにして</td>
			<td>TGACGAACGTGGTTAGTCGT</td>
		</tr>
<tr>
<td>前 53_R</td>
			<td>リバース</td>
			<td>GGCATAACGTCCTAGTCACA</td>
		</tr>
<tr>
<td>前 59_F</td>
			<td>楽しみにして</td>
			<td>GCGAGTGGTATTCGCTTAGT</td>
		</tr>
<tr>
<td>前 59_R</td>
			<td>リバース</td>
			<td>GTTACCCTCGACGATTGCGT</td>
		</tr>
<tr>
<td>前 66_F</td>
			<td>楽しみにして</td>
			<td>TCGGTTTGGCAACGTCGGGC</td>
		</tr>
<tr>
<td>前 66_R</td>
			<td>リバース</td>
			<td>GACTAGGGAGATGCCGGCGA</td>
		</tr>
<tr>
<td>前 69_F</td>
			<td>楽しみにして</td>
			<td>CGACTCAGCCCCGAGATTT</td>
		</tr>
<tr>
<td>前 69_R</td>
			<td>リバース</td>
			<td>ATACAAGCAAACATAGACGGAA</td>
		</tr>
<tr>
<td>前 84_F</td>
			<td>楽しみにして</td>
			<td>GACAGTGGTGAGGTTTCAA</td>
		</tr>
<tr>
<td>前 84_R</td>
			<td>リバース</td>
			<td>ACTGGCGTTACCTTGTCTA</td>
		</tr>
<tr>
<td>前 89_F</td>
			<td>楽しみにして</td>
			<td>GAACAGTGCTCGCAGTCTAT</td>
		</tr>
<tr>
<td>前 89_R</td>
			<td>リバース</td>
			<td>GACAGCGTAAACATCGCGGT</td>
		</tr>
<tr>
<td>前 126_F</td>
			<td>楽しみにして</td>
			<td>GGTACATTCGTGTCGATTT</td>
		</tr>
<tr>
<td>前 126_R</td>
			<td>リバース</td>
			<td>TAAACGAAAAAACCACGTAC</td>
		</tr>
</tbody></table>表 1: 遺伝子型に使用されるマイクロ サテライト プライマー シーケンスワタアブラムシ nerii20.
<table fo:keep-together.within-page="1"><tbody>
<tr>
<td>ターゲット</td>
			<td>サンプル</td>
			<td>Ct の意味</td>
			<td>Ct 標準開発</td>
			<td>ΔCt</td>
			<td>平均 ΔCt</td>
			<td>ΔΔCt</td>
			<td>RQ=2^(-ΔΔCt)</td>
			<td>RQ SEM</td>
		</tr>
<tr>
<td>ef1a</td>
			<td>1.1</td>
			<td>22.59</td>
			<td>0</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>1.2</td>
			<td>20.31</td>
			<td>0</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>1.3</td>
			<td>20.36</td>
			<td>0.226</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>1.4</td>
			<td>20.27</td>
			<td>0.036</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>1.5</td>
			<td>20.55</td>
			<td>0.003</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>1.6</td>
			<td>20.52</td>
			<td>0.245</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>2.1</td>
			<td>20.49</td>
			<td>0.082</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>2.2</td>
			<td>19.86</td>
			<td>0.033 お</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>2.3</td>
			<td>20.19</td>
			<td>0.037</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>2.4</td>
			<td>19.67</td>
			<td>0.058</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>2.5</td>
			<td>20.25</td>
			<td>内径 0.188</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>2.6</td>
			<td>18.16</td>
			<td>0.089</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>3.1</td>
			<td>20.93</td>
			<td>0.157</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>3.2</td>
			<td>20.22</td>
			<td>0.003</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>3.3</td>
			<td>20.44</td>
			<td>0.039</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>3.4</td>
			<td>20.91</td>
			<td>0.559</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>3.5</td>
			<td>20.63</td>
			<td>0.017</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>ef1a</td>
			<td>3.6</td>
			<td>20.3</td>
			<td>0.135</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>1.1</td>
			<td>24.6</td>
			<td>0.173</td>
			<td>2.01</td>
			<td></td>
			<td>0</td>
			<td>1</td>
			<td></td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>1.2</td>
			<td>24.25</td>
			<td>0.019</td>
			<td>3.94</td>
			<td>2.975</td>
			<td>0</td>
			<td>1</td>
			<td>0</td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>1.3</td>
			<td>24.79</td>
			<td>0.04</td>
			<td>4.43</td>
			<td></td>
			<td>0</td>
			<td>1</td>
			<td></td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>1.4</td>
			<td>25.23</td>
			<td>0.285</td>
			<td>4.96</td>
			<td>4.695</td>
			<td>0</td>
			<td>1</td>
			<td>0</td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>1.5</td>
			<td>24.6</td>
			<td>0.103</td>
			<td>4.05</td>
			<td></td>
			<td>0</td>
			<td>1</td>
			<td></td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>1.6</td>
			<td>25.08</td>
			<td>0.033 お</td>
			<td>4.56</td>
			<td>4.305</td>
			<td>0</td>
			<td>1</td>
			<td>0</td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>2.1</td>
			<td>27.52</td>
			<td>0.155</td>
			<td>7.03</td>
			<td></td>
			<td>5.019033762</td>
			<td>0.03084042</td>
			<td></td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>2.2</td>
			<td>27.23</td>
			<td>0.061</td>
			<td>7.37</td>
			<td>7.2</td>
			<td>3.428355679</td>
			<td>0.092888533</td>
			<td>0.031024057</td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>2.3</td>
			<td>27.18</td>
			<td>0.058</td>
			<td>6.99</td>
			<td></td>
			<td>2.56158174</td>
			<td>0.169389724</td>
			<td></td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>2.4</td>
			<td>27.45</td>
			<td>0</td>
			<td>7.78</td>
			<td>7.385</td>
			<td>2.820764967</td>
			<td>0.141535419</td>
			<td>0.013927153</td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>2.5</td>
			<td>27.44</td>
			<td>0.032</td>
			<td>7.19</td>
			<td></td>
			<td>3.138956897</td>
			<td>0.113521944</td>
			<td></td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>2.6</td>
			<td>28</td>
			<td>0</td>
			<td>9.84</td>
			<td>8.515</td>
			<td>5.284272079</td>
			<td>0.025661119</td>
			<td>0.043930413</td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>3.1</td>
			<td>27.23</td>
			<td>0.143</td>
			<td>6.3</td>
			<td></td>
			<td>4.292437371</td>
			<td>0.051032588</td>
			<td></td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>3.2</td>
			<td>27.05</td>
			<td>0.088</td>
			<td>6.83</td>
			<td>6.565</td>
			<td>2.891234282</td>
			<td>0.134788164</td>
			<td>0.041877788</td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>3.3</td>
			<td>27.45</td>
			<td>0.109</td>
			<td>7.01</td>
			<td></td>
			<td>2.578145722</td>
			<td>0.167456035</td>
			<td></td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>3.4</td>
			<td>27.58</td>
			<td>0.019</td>
			<td>6.67</td>
			<td>6.84</td>
			<td>1.709038085</td>
			<td>0.305863936</td>
			<td>0.069203951</td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>3.5</td>
			<td>27.06</td>
			<td>0.067 倍</td>
			<td>6.43</td>
			<td></td>
			<td>2.384498984</td>
			<td>0.191511246</td>
			<td></td>
		</tr>
<tr>
<td>興味の遺伝子</td>
			<td>3.6</td>
			<td>27.36</td>
			<td>0</td>
			<td>7.06</td>
			<td>6.745</td>
			<td>2.513723938</td>
			<td>0.175103043</td>
			<td>0.008204101</td>
		</tr>
</tbody></table>表 2: qPCR ΔΔC の計算t候補遺伝子の検証します。候補遺伝子発現 ef1a を基準にして計算されます (図 6)。1.1 1.6 表す 6 生物複製処理; 下のサンプルします。2.1 2.6 表す 6 生物複製処理 1; 下のサンプルします。3.1 3.6 表す 6 生物学的治療法 2 下で複製をサンプリングします。Ct標準の開発は、3 つの技術的な複製から計算されます。

Watch this Scientific Journal Video about Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions at JoVE.com

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

アブラムシワタアブラムシ nerii付いているバシクルモン家族 (Apocyanaceae) で強く擁護した植物の定着植物昆虫の相互作用を研究する多くの機会を提供しています。ここでは、我々 の文化と生成分子の解析植物とアブラムシの保守のためのプロトコルのシリーズの紹介とA. nerii用プロファイルー データ。

生物学的文化とワタアブラムシ neriiの遺伝子発現を測定を維持する: 植物・昆虫間の相互作用の非モデル系

Aphids are excellent experimental models for a variety of biological questions ranging from the evolution of symbioses and the development of polyphenisms ...

maintaining-biological-cultures-measuring-gene-expression-aphis-nerii

Nanyang Technological University

Research

JoVE Journal

Genetics

Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

7.5K ビュー.  Vanderbilt University. アブラムシワタアブラムシ nerii付いているバシクルモン家族 (Apocyanaceae) で強く擁護した植物の定着植物昆虫の相互作用を研究する多くの機会を提供しています。ここでは、我々 の文化と生成分子の解析植物とアブラムシの保守のためのプロトコルのシリーズの紹介とA. nerii用プロファイルー データ。

ビデオ: Maintaining Biological Cultures and Measuring Gene Expression in Aphis nerii: A Non-model System for Plant-insect Interactions

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

ヒト初代細胞培養物における概日時計遺伝子発現やホルモン分泌のパラレル測定

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

遺伝学

Perturbations of Circulating miRNAs in Irritable Bowel Syndrome Detected Using a Multiplexed High-throughput Gene Expression Platform

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in a single reaction. The miRNA assay counts transcripts by directly imaging and digitally counting miRNA molecules that are labeled with color-coded fluorescent barcoded probe sets (a reporter probe and a capture probe). Barcodes are hybridized directly to mature miRNAs that have been elongated by ligating a unique oligonucleotide tag (miRtag) to the 3&#39; end. Reverse transcription and amplification of the transcripts are not required. Reporter probes contain a sequence of six color positions populated using a combination of four fluorescent colors. The four colors over six positions are used to construct a gene-specific color barcode sequence. Post-hybridization processing is automated on a robotic prep station. After hybridization, the excess probes are washed away, and the tripartite structures (capture probe-miRNA-reporter probe) are fixed to a streptavidin-coated slide via the biotin-labeled capture probe. Imaging and barcode counting is done using a digital analyzer. The immobilized barcoded miRNAs are visualized and imaged using a microscope and camera, and the unique barcodes are decoded and counted. Data quality control (QC), normalization, and analysis are facilitated by a custom-designed data processing and analysis software that accompanies the assay software. The assay demonstrates high linearity over a broad range of expression, as well as high sensitivity. Sample and assay preparation does not involve enzymatic reactions, reverse transcription, or amplification; has few steps; and is largely automated, reducing investigator effects and resulting in high consistency and technical reproducibility. Here, we describe the application of this technology to identifying circulating miRNA perturbations in irritable bowel syndrome.

We describe the use of a multiplexed high-throughput gene expression platform that quantitates gene expression by barcoding and counting molecules in biological substrates without the need for amplification. We used the platform to quantitate microRNA (miRNA) expression in whole blood in subjects with and without irritable bowel syndrome.

過敏性腸症候群におけるmiRNA循環の摂動は多重化ハイスループット遺伝子発現プラットフォームを使用して検出します

The gene expression platform assay allows for robust and highly reproducible quantification of the expression of up to 800 transcripts (mRNA or miRNAs) in ...

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

内部標準でFACSおよび定量PCRを用いた単一細胞遺伝子発現プロファイリング

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

サンプル準備およびゼブラフィッシュから RNASeq による遺伝子発現データの解析

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The mechanisms of GA and ABA signaling can be interrogated by introducing reporter gene constructs into aleurone cells via particle bombardment, with the resulting transient expression measured using enzyme assays. An improved protocol is reported that partially automates and streamlines the grain homogenization step and the enzyme assays, allowing significantly more throughput than existing methods. Homogenization of the grain samples is carried out using an automated tissue homogenizer, and GUS (&#946;-glucuronidase) assays are carried out using a 96-well plate system. Representative results using the protocol suggest that phospholipase D activity may play an important role in the activation of HVA1 gene expression by ABA, through the transcription factor TaABF1.

An improved protocol is presented for the measurement of transient gene expression from reporter constructs in barley aleurone cells after particle bombardment. The combination of automated grain grinding with 96-well plate enzyme assays provides high throughput for the procedure.

スループットを向上させると衝撃されたオオムギ糊粉層における遺伝子発現を測定

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

効率的な生産とモデル システムの動脈分布に CRISPR 識別/Cas9 生成遺伝子のノックアウト

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

制御、細胞間相互作用における遺伝子発現パターンを分析して光遺伝学的方法

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene switches requires the transfer of two therapeutic expression units concurrently into a single cell, the practical application of the system for gene therapy is limited. To simplify the transgene expression system, we generated a gene switch designated as pEUI(+) encompassing a complete set of transgene expression modules in a single vector. Comprising of the GAL4 DNA-binding domain and modified EcR (GvEcR), a minimal VP16 activation domain fused with a GAL4 DNA-binding domain, as well as a modified Drosophila ecdysone receptor (EcR), the newly developed singular gene switch is highly responsive to the administration of a chemical inducer in a time- and dosage-dependent manner. The pEUI(+) vector is a potentially powerful tool for improving the control of transgene expression in both biological research and pre-clinical studies. Here, we present a detailed protocol for modulation of a transient and stable transgene expression using pEUI(+) vector by the treatment of tebufenozide (Teb). Additionally, we share important guidelines for the use of Teb as a chemical inducer.

Here, we present a protocol for the modulation of transgene expression using pEUI(+) singular gene switch by tebufenozide treatment.

脱皮ホルモン受容体を介した特異遺伝子スイッチ意図的な堅牢性、可逆性、ごくわずかな水漏れしたためと遺伝子発現の

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

探索ルート マイクロバイ: 土、根、およびルート Endosphere から細菌群集のデータを抽出

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

多重遺伝子型及び質量分析法と臨床コホート研究でテスト候補遺伝子

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...