この作品は、モンサント社のトウモロコシ Rootworm 知識研究プログラムからの助成金によって支えられた、NCSU から MDL に番号 AG/1005 (MDL と YC) してスタートアップ資金を与えます。(MDL) に番号 MCB 1244772 を付与する FC は、モンサント社のトウモロコシ Rootworm 知識研究プログラム (AG/1005)、全米科学財団からの助成金によって支えられました。著者は競争の興味を宣言しません。FC と MDL の考案設計実験;FC、PW、SP で行う実験。FC と MDL の分析結果FC、NG、MDL は原稿を書いていた。WCR のスクリーニングでの専門的な支援、テレサ o ' leary、ウィリアム Klobasa とステファニーと感謝します。我々 はまた飼育プロトコルを提供するラボのコロニーを確立する卵の出荷のため博士のウェイドのフランス語 (農務省、北中央農業研究所、ブルッキングズ, サウスダコタ) を感謝します。

1. コロニー レベル WCR 大人の飼育
<ol><li>WCR 大人の十分な量を取得 (500-1,000) 信頼性の高い会社や研究所から (例のための参照材料表) し、30 cm3ケージに配置。 注: 非休眠株の使用は強くお勧めします。</li>
	<li>WCR 人工飼料メーカーのプロトコル、次を準備し、38 オンスの容器に 1 cm 厚層を注ぐ (材料の表を参照してください)。混合した後、2 ヶ月未使用ダイエット 4 ° C で保存します。</li>
	<li>大人の食事 (10-15 g) とペトリ皿 (100 × 15 mm) を 30 cm3ケージに入れます。低または乾燥食品より多くの食事を追加します。</li>
	<li>水の源として水、コットン ロール (6"x 3/8")、提供する場所に保持するために使用する綿球で覆われているとフラスコ (300 mL) を置きます。低実行するときは、水を変更します。</li>
	<li>26 ° C 湿度 60% で WCR を維持し、14:10 光インキュベーターを飼育昆虫のサイクルします。</li>
</ol>2. 胚のコレクションおよび配置
<ol><li>シャーレの滅菌 100 × 15 mm2水 1% 寒天を用いた卵コレクション区域を作る。寒天が固化した後、4 ° C で保存します。</li>
	<li>最近置かれた卵を収集するには、(図 1 a) 寒天培地の表面に寒冷紗 (サイズへのカットそれぞれ) の 4 層に続いて、フィルター ペーパーの単一の層を配置します。 メモ: 寒冷紗を使用できます、漂白剤で洗浄し、オートクレーブ養生した後、初めて使用するためこれを行う必要はありません。フィルター紙は再利用されないことし、滅菌する必要はありません。</li>
	<li>できるだけ (仕事日の終わり) の日に遅く WCR ケージに卵コレクション プレートを置き、アルミ箔のテントで覆います。一晩おきます。 ケージに夜遅くコレクション区域を配置するラボのスタッフを必要とせずマイクロインジェクションの若い卵のコレクションを進め注: しなくてもライト 10 から 12 時 (14 時間) 遅れ産卵。</li>
	<li>卵コレクション区域約 8 または 9 を削除します。</li>
	<li>鉗子を使用すると、時間と水のビーカー (500 mL) の場所で寒冷紗の 1 層を拾います。(図 1 b と 1 C) を優しく攪拌によってチーズの卵を洗います。</li>
	<li>電球ピペットを使用して卵を水の別のビーカー (500 mL) に転送。卵をきれいにする 2 〜 3 倍を繰り返します。</li>
	<li>水繰越額を最小限に抑えながら、フィルター紙 (図 1) の上に卵を転送するのに電球ピペットを使用します。</li>
	<li>しっかりと確認するスライドにスライド ガラスとテープと同じ幅の短冊状に黒濾紙をカット紙産むフラット (図 2 a)。 注: 黒濾紙は、反射する光の量を減らすことによって顕微鏡下で可視性を向上させるのに役立ちます。</li>
	<li>非毒性接着剤の適用 (例えば材料の表を参照) 細い線 (図 2 b) でろ過紙。接着剤でコーティングされた卵を避けるために卵の直径よりも薄く、接着をしてください。</li>
	<li>軽く接着剤のラインに彼らのフィルター ペーパーから WCR 卵 1 つずつを移動するのに細かいブラシを使用します。接着剤で産卵を乾く前にしようし、それぞれの卵の間に少なくとも 1 つの卵の距離を保ちます。</li>
	<li>1 ろ紙スライド (図 3) に複数行の卵を置くことによって注入効率を最大化します。</li>
	<li>すべての卵をフィルター紙の上にレイアウト後のインジェクションの前にすべての接着剤が乾くまで待ちます。 注: 生殖変換と編集 CRISPR/Cas9 を介したゲノム、注入すべて胚正午 (12) 19 h 以上古い最古の胚があるよう。しかし、RNAi のため制限はありません時間、WCR で dsRNA セル間を移動することができます。実際には、RNAi のため高齢化胚がよりよい生存率で可能性があります。</li>
</ol>3. 保有するプラスミド Dna と注射針の準備
<ol><li>エンドトキシン フリーの商業を使用して高品質スーパー プラスミッド DNA の準備キット (例については材料の表を参照してください)。</li>
	<li>注射液の準備するには、各プラスミドの濃度をチェックし、ヘルパー プラスミド (250 ng/μ L 最終濃度)、ドナー プラスミド (750 ng/μ L 最終濃度)、フェノールレッド バッファー (最終濃度 20%)、結合します。渦ソリューションを簡単に、針を詰まらせることができる粒子の沈殿物に最大速度で 3 分間遠心します。</li>
	<li>ホウケイ酸ガラスの針 (外径内径 0.58 mm、10 cm 長さ 1 mm) を引く炎/ブラウン タイプのマイクロ ピペットの引き手を使用して。ストレージ、シャーレなどの明確なコンテナーの両面粘着テープで引っ張ら針を固定します。 注: マイクロ ピペットの引き手システムの使用設定があった: 熱 335、フィルを = = 4、ヴェル = 40、デル = 200、PUL = 100。</li>
	<li>バックフィル マイクロ ローダーを使用して、注射針ヒント (材料の表を参照) をピペット 0.5 - 1.0 μ L をテーパー エンドの近くの針の内部。発生した場合は、針の先端から泡を削除します。</li>
	<li>持針器に注射針を挿入します。</li>
	<li>優しく、鉗子の罰金ペアを破壊または優しく触れる/ドラッグ coverslip またはガラス スライドの先端の針の先端を開きます。 注: まだ出てくる注射ミックスを可能にする最小の開口部を持っていること (小さな開口部は一般的に高い注入圧力を必要と)。ベベル針は、彼らは既に開いているのでこの手順を必要としません。</li>
</ol>4. マイクロインジェクションと投与後のケア
<ol><li>細かいペイント ブラシを使用して、慎重に滅菌水で 1-3 個の卵の表面を湿らせる針を挿入、薬液を注入します。表面が乾く前に、それぞれの卵を注入します。成功のインジェクションを示す注入中に卵中の赤い色の少量を外観を探します。クラッシュまたは破損、空を見て、またはそれ以外の場合、注入することはできませんのすべての卵を削除します。 注: 射出圧力によって異なりますが針穴のサイズに基づいて、10-13 psi は良い出発点です。</li>
	<li>すべての卵が注入された後は、スライド ガラスからフィルター用紙を取り除きます。</li>
	<li>(上記) 1% 寒天料理の表面にフィルター紙を置きます。</li>
	<li>料理を密封し、12 日間インキュベーターを飼育 26 ° C 昆虫を置くプラスチック パラフィン フィルムを使用します。 注: パラフィン フィルムの料理の間に金型のクロス汚染を防ぐ助け。しかし、カビや細菌の増殖を防ぐために他の治療法は、注入された胚の生存率を減らすことができます。</li>
</ol>5. 培養と胚の孵化
<ol><li>孵化胚 12 日後注入をチェックを開始し、次の 2 週間毎日続けてください。</li>
	<li>発芽トウモロコシと土飼育箱に細かいブラシを使用して、幼虫を転送します。 注: 金型は、寒天料理で成長可能性がありますが、ほとんどの場合幼虫が孵化まだ。それらすべてをキャッチする金型内で慎重に幼虫を探します。いくつか幼虫は皿の蓋に移動可能性があり、結露にはまり込む可能性があります。</li>
	<li>26 ° C 以下の飼育方法25標準 WCR で幼虫を育てます。</li>
</ol>

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G., Roth, S., Sander, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tribolium+embryogenesis:+a+SEM+study+of+cell+shapes+and+movements+from+blastoderm+to+serosal+closure.">Tribolium embryogenesis: a SEM study of cell shapes and movements from blastoderm to serosal closure.</a> Dev Genes Evol. 210 (4), 167-179 (2000).</li><li>Handler, A. M., James, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Insect transgenesis: methods and applications. , (2000).</li><li>Lorenzen, M. D., 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=piggyBac-based+insertional+mutagenesis+in+Tribolium+castaneum+using+donor/helper+hybrids.">piggyBac-based insertional mutagenesis in Tribolium castaneum using donor/helper hybrids.</a> Insect Mol Biol. 16 (3), 265-275 (2007).</li><li>Gilles, A. F., Schinko, J. B., Averof, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+CRISPR-mediated+gene+targeting+and+transgene+replacement+in+the+beetle+Tribolium+castaneum.">Efficient CRISPR-mediated gene targeting and transgene replacement in the beetle Tribolium castaneum.</a> Development. 142 (16), 2832-2839 (2015).</li><li>Phuc, H. K., 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=Late-acting+dominant+lethal+genetic+systems+and+mosquito+control.">Late-acting dominant lethal genetic systems and mosquito control.</a> BMC Biol. 5, 11 (2007).</li><li>Horn, C., Wimmer, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgene-based,+embryo-specific+lethality+system+for+insect+pest+management.">A transgene-based, embryo-specific lethality system for insect pest management.</a> Nat Biotechnol. 21 (1), 64-70 (2003).</li></ol>

著者が明らかに何もありません。

これはマイクロインジェクション precellular WCR 胚生殖細胞への変換を行うための重要なプロセスのためのベストプラクティスを確立する最初のプロトコルで RNAi を目的として二本の WCR のマイクロインジェクション報告9がずっと、および/またはこの種の編集 CRISPR/Cas9 ゲノム。WCR 胚の正常なマイクロインジェクションは、変換効率は、多くの要因に依存しています。後述は?...

西部のトウモロコシ rootworm (WCR)、Diabrotica virgifera virgifera、トウモロコシ1の重要な害虫であります。興味深いことに、WCR のコントロールを克服するよう措置最も農業害虫よりも急速に以来、彼らだけでなく適応だけでなく、生理学的行動2,3,4,5, 6. 過去 10 年間、RNA 干渉 (RNAi) 強力な機能ゲノム ツール WCR7、8、潜在的な制御法として研究されている、遺伝子機能9を研究手段としても使用されています。しかし、RNAi は頻繁に他の種における二本鎖 rna (dsRNA) によって実行される、インジェクション ベース RNAi は WCR にまれであります。実際、dsRNA の WCR9にマイクロインジェクションによるRNAi の報告は少ないがあります。理由は母11に dsRNA を供給することにより胚性効果の研究を許しさえ WCR が dsRNA7,10の遺伝子ノックダウンを介して摂取の高レベルに得ることができます。このメソッドには、WCR 機能ゲノム解析によるRNAi 優秀な主題が、それはこの種の胚のインジェクションの方法論の開発の進捗状況を鈍化しています。
RNAi のパワーにもかかわらず、いくつかの欠点があります。たとえば、すべての遺伝子は RNAi に均等に対応します。この変動より困難なゲノム機能アッセイの結果を解釈することができます。また、RNAi は一時的なもの、遺伝的突然変異を生成しません。その一方で、生殖細胞変換、機能ゲノムの他のツールはマーク付きの転位因子のゲノム12,13にを介して挿入変異を生成できます。これは生殖細胞変換を長期的な遺伝学的研究で使用するため突然変異系統を作成するための優れたツールになります。変換は、変異ゲノム14遺伝子の機能的なコピーを提供することにより、突然変異を救出するも使用できます。さらに、生殖細胞変換はさまざまな分子遺伝学的手法の礎です。ノックアウトや遺伝子機能を救出、に加えてエンハンサー トラップ15、遺伝子トラップ16、Gal4 ベースの異所性発現17、およびゲノム変異18の生殖細胞への変換を使用できます。
最近では、高度なゲノムの編集を有効にするツールが開発されています。これらのツールでは、転写 Activator-Like エフェクター核酸 (TALEN) CRISPR/Cas9-ヌクレアーゼ システム19,20をご利用など。これらの新しいツールの利点は、彼らは研究者のほぼすべてのゲノム内のほぼすべての場所で二本鎖 DNA 切断を誘導する能力を提供することです。どちらか非相同末端結合、削除、または目標とされた遺伝子の挿入を導入することができます、通したり、相同性監督の修理エンジニア リング構造が存在することができますを触媒する誘導、一度これらの壊れ目を修理すること、全体の遺伝子や遺伝領域21,22の交換。ただし、これらのメソッドには、DNA、RNA および蛋白質の非常に若い胚へのマイクロインジェクションが必要があります。
重要なは、RNAi、生殖のために使用される蛋白質や核酸用二本とは異なり、変換およびゲノムの編集が、細胞膜を容易に越えることはできません。したがって、プラスミド Dna や Mrna、タンパク質のマイクロインジェクションは胞胚期 (すなわち昆虫胚 cellularizes 前に) 行われなければなりません。これは注射のタイミングを重要な要因になります。たとえば、キイロショウジョウバエ胚は卵を産む12後 2 時間以内に注入する必要があります。したがって、WCR の正常胚マイクロインジェクションのプロトコルの開発は、precellular 胚の十分な量を収集するための最良の方法だけでなく、女性の卵が産卵に最適な条件に考慮に入れなければなりません。
WCR 生物学を分子遺伝学的ツールの広い範囲をもたらすべく収集と precellular WCR 胚マイクロインジェクション法の開発が必要です。ここで我々 は WCR 研究に変換ベースの技術を使用して他人を助けるためのヒントとトリックの詳細な手順を提供します。これらのテクニックには WCR 機能ゲノム研究用トランスジェニック技術を拡張すると、に加えて、遺伝子ドライブ23,24経済的に重要なこれでテストするなど強力な新しい害虫対策も有効にします。害虫。

<table border="0" cellpadding="0" cellspacing="0" width="1155">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Qualitative Filter Paper (Black)</td>
			<td style="width:189px;">Ahlstrom</td>
			<td style="width:287px;">8613-0900</td>
			<td style="width:339px;">For egg microinjection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 oz Containers&#160;</td>
			<td style="width:189px;">Anny&#39;s Plastic Tableware</td>
			<td>ASET101</td>
			<td>Egg container</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drosophila Agar Type II</td>
			<td style="width:189px;">Apex</td>
			<td style="width:287px;">66-103</td>
			<td>Substrate for WCR egg-laying dish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Featherweight Forceps&#160;</td>
			<td>Bioquip</td>
			<td style="width:287px;">4748</td>
			<td>Handling larvae and pupae&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clorox Regular-Bleach1</td>
			<td style="width:189px;">Clorox</td>
			<td style="width:287px;">44600-30770</td>
			<td>Wash corn and dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trucker&#39;s Favorite Yellow</td>
			<td>Coor Farm Supply</td>
			<td>502</td>
			<td>Corn for feeding WCR</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Microinjection System (Homemade)</td>
			<td style="width:189px;">NA</td>
			<td style="width:287px;">Parts &#38; instructions available upon request</td>
			<td>Controls injection pressure (0-20 psi)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Elmer&#39;s Non-toxic Glue</td>
			<td>Elmer&#39;s Products, Inc.</td>
			<td style="width:287px;">E304</td>
			<td>Glue eggs on filter paper</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">epTIPS Microloader Tips</td>
			<td style="width:189px;">Eppendorf</td>
			<td style="width:287px;">C2554691</td>
			<td>Backfilling needle loading tips</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon Tissue Culture Dishes</td>
			<td style="width:189px;">Falcon</td>
			<td>25383-103</td>
			<td>Sprouting corn /150 x 25 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisherbrand&#160;Quantitative-Grade Filter Paper Circles</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td>S47576C</td>
			<td>Making agar dish for egg-lay/9cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Sparkleen</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td style="width:287px;">04-320-4</td>
			<td>Wash dishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Plain Microscope Slides</td>
			<td style="width:189px;">Fisher Scientific</td>
			<td style="width:287px;">12-549-3</td>
			<td>Holding filter paper and eggs for microinjection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Western Corn Rootworm w/o Pollen Substitute</td>
			<td style="width:189px;">Frontier Agricultural Sciences&#160;</td>
			<td>F9766B</td>
			<td>WCR adult artificial diet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cotton Balls, Large</td>
			<td>Genesee Scientific</td>
			<td>51-101</td>
			<td>Close the flask</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Globe Scientific 3.0 mL Small Bulb Transfer Pipettes</td>
			<td>Globe Scientific</td>
			<td>137035</td>
			<td>Collect and transfer eggs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica M165 FC Fluorescence Stereomicroscope&#160;</td>
			<td style="width:189px;">Leica</td>
			<td>M165 FC&#160;</td>
			<td>WCR screening</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DsRed Filter Set for Fluorescence Stereomicroscope</td>
			<td style="width:189px;">Leica</td>
			<td>DSR</td>
			<td>Excitation filter: 510-560 nm, emission filter: 590-650 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGFP filter</td>
			<td style="width:189px;">Leica</td>
			<td>GFP2</td>
			<td>Excitation filter: 440&#8211;520 nm, emission filter: 510 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BugDorm</td>
			<td>MegaView Science</td>
			<td>DP1000_5P</td>
			<td>Cage for adult colony</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Miniature Paint Brush</td>
			<td style="width:189px;">MyArtscape</td>
			<td>MAS-102-MINI</td>
			<td>Liner 2/0&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Joystick Micromanipulator</td>
			<td style="width:189px;">Narishige</td>
			<td style="width:287px;">MN-151</td>
			<td>Micromanipulator and needle holder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">Microscope XY Stage</td>
			<td style="width:189px;">Olympus</td>
			<td style="width:287px;">265515</td>
			<td style="width:339px;">Microscope stage (adapted for use with stereoscope)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Grade 90 Cheesecloth</td>
			<td style="width:189px;">Online Fabric Store</td>
			<td>CHEE90</td>
			<td>For egg-lay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic paraffin film&#160;</td>
			<td style="width:189px;">Pechiney Plastic Packaging</td>
			<td>PM-996</td>
			<td>Seal agar dish after microinjection/Roll size 4 in. x 125 ft</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Percival Incubator</td>
			<td style="width:189px;">Percival&#160;</td>
			<td style="width:287px;">I41VLH3C8</td>
			<td>WCR growing chamber (insect rearing incubator)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Narrow Mouth Erlenmeyer Flasks</td>
			<td style="width:189px;">VWR</td>
			<td>4980-300-PK</td>
			<td>Water container for adult colony&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Griffin Low Form Beakers</td>
			<td style="width:189px;">VWR</td>
			<td>1000-600-PK</td>
			<td>For egg wash</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Braided Cotton Rolls</td>
			<td>Richmond Dental Cotton Co.</td>
			<td>605-3599</td>
			<td>Use in water supply for adult colony&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dishes (35x10mm)</td>
			<td>SIGMA</td>
			<td>CLS430588-500EA</td>
			<td>For diet plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenol Red</td>
			<td>Sigma</td>
			<td style="width:287px;">143-78-8</td>
			<td>Microinjection buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laser-based Micropipiette Puller</td>
			<td>Sutter Instrument</td>
			<td>P-2000/G</td>
			<td>Needle puller/Heat = 335, FIL = 4, VEL = 40, DEL = 200, PUL = 100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Premium Topsoil</td>
			<td>The Scotts Company</td>
			<td style="width:287px;">71130758</td>
			<td>Soil for cron growing</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reloc Zippit 2 Mil Zipper Bags</td>
			<td>United States Plastic Corporation</td>
			<td>48342</td>
			<td>Bag agar dish after microinjection/5x7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dishes (100x15m)</td>
			<td style="width:189px;">VWR</td>
			<td style="width:287px;">89038-968</td>
			<td>Making agar dish for egg-lay/ 100 x 15 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 oz Containers</td>
			<td style="width:189px;">Webstaurantstore</td>
			<td>128E506</td>
			<td>WCR single pair mating chamber&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">38 oz Containers</td>
			<td style="width:189px;">Webstaurantstore</td>
			<td>128NC888</td>
			<td>Larvae rearing box/38 oz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChoiceHD 16 oz. Microwavable Translucent Plastic Deli Container</td>
			<td style="width:189px;">Webstaurantstore</td>
			<td>128HRD16</td>
			<td>Larvae rearing box/16 oz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Microinjection Scope&#160;</td>
			<td style="width:189px;">Wild Heerbrugg</td>
			<td style="width:287px;">WILD-M8</td>
			<td style="width:339px;">Microinjection scope outfited with an XY stage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100F-4</td>
			<td>Microinjection needles</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:343px;">Adult Western Corn Rootworms</td>
			<td style="width:189px;">North Centeral Aqricultural reasearch Laboratory</td>
			<td style="width:287px;">Non-diapase strain</td>
			<td>Request from Dr. Bryan Wade French&#39;s lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:343px;">Plasmid DNA Midi Kit</td>
			<td style="width:189px;">Qiagen</td>
			<td>12143</td>
			<td>Purification of injection-ready plasmid DNAs</td>
		</tr>
	</tbody>
</table>

microinjection of western corn rootworm, diabrotica virgifera virgifera, embryos for germline transformation, or crispr/cas9 genome editing

西部のトウモロコシ rootworm (WCR) トウモロコシの重要害虫であり害虫管理戦略に迅速に適応する能力があることが知られています。RNA 干渉 (RNAi) WCR 生物学を勉強するための強力なツールであることを証明している、それはその限界を持っています。具体的には、RNAi 自体は一時的なもの (すなわちわけで関連する表現型の長期的なメンデル遺伝)、それはターゲット遺伝子の DNA 順序を知る必要があり、。後者ために制限があります興味の表現型が悪い、節約またはも新規の遺伝子によって制御される場合便利なターゲットの識別に挑戦するが、不可能ではない場合。WCR のゲノム] ツールボックスのツールの数は、安定した突然変異系統を作成し、WCR ゲノムのシーケンスに依存しない調査を有効にするために使用できる手法の開発が拡大する必要があります。ここで、収集し、核酸 precellular WCR 胚を microinject するために使用方法を詳しく説明します。ここ記載されているプロトコルは、トランスジェニック WCR の創出を目指したが、一方 CRISPR/Cas9-ゲノム編集も実行でした唯一の違いは、胚に注入液の組成と同じプロトコルを使用しています。

このプロトコルを開発するときの重要な考慮事項は、マイクロインジェクションの時に萌芽期の開発の段階をだった。具体的には、生殖細胞系列変換は萌芽期の cellularization の前に dna マイクロインジェクションを必要です。これは、Dna が細胞膜を容易に通過できないためにです。WCR 胚の dechorionation は本質的にすべての胚の完全性に責任がある保護膜を溶解するため...

Watch this Scientific Journal Video about Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing at JoVE.com

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

マイクロインジェクション ウエスタン トウモロコシ Rootworm、 Diabrotica virgifera virgifera胚の生殖細胞への変換、または CRISPR/Cas9 ゲノム編集

ここで rootworm 胚生殖細胞変換や CRISPR/Cas9-ゲノム編集など機能ゲノム試金の実行の目的のため収集、マイクロインジェクション precellular 西部のトウモロコシのためのプロトコルは提示します。

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although ...

microinjection-western-corn-rootworm-diabrotica-virgifera-virgifera

Research

JoVE Journal

Bioengineering

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

7.3K ビュー.  North Carolina State University. ここで rootworm 胚生殖細胞変換や CRISPR/Cas9-ゲノム編集など機能ゲノム試金の実行の目的のため収集、マイクロインジェクション precellular 西部のトウモロコシのためのプロトコルは提示します。

ビデオ: Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

のためのウイルス粒子<emインビボ&gt;</em&gt;腫瘍イメージング

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

生物工学

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

マイクロインジェクションアッセイをキズや<em&gt;インビボ</em&gt;表皮の局在は、応答記者傷<em&gt;ショウジョウバエ</em&gt;胚。

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

Efficient Generation and Editing of Feeder-free IPSCs from Human Pancreatic Cells Using the CRISPR-Cas9 System

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted for research in regenerative medicine and are currently in clinical trials for eye diseases, diabetes, heart diseases, and other disorders. The potential to differentiate into specialized cell types coupled with the recent advances in genome editing technologies including the CRISPR/Cas system have provided additional opportunities for tailoring the genome of iPSC for varied applications including disease modeling, gene therapy, and biasing pathways of differentiation, to name a few. Among the available editing technologies, the CRISPR/Cas9 from Streptococcus pyogenes has emerged as a tool of choice for site-specific editing of the eukaryotic genome. The CRISPRs are easily accessible, inexpensive, and highly efficient in engineering targeted edits. The system requires a Cas9 nuclease and a guide sequence (20-mer) specific to the genomic target abutting a 3-nucleotide &#34;NGG&#34; protospacer-adjacent-motif (PAM) for targeting Cas9 to the desired genomic locus, alongside a universal Cas9 binding tracer RNA (together called single guide RNA or sgRNA). Here we present a step-by-step protocol for efficient generation of feeder-independent and footprint-free iPSC and describe methodologies for genome editing of iPSC using the Cas9 ribonucleoprotein (RNP) complexes. The genome editing protocol is effective and can be easily multiplexed by pre-complexing sgRNAs for more than one target with the Cas9 protein and simultaneously delivering into the cells. Finally, we describe a simplified approach for identification and characterization of iPSCs with desired edits. Taken together, the outlined strategies are expected to streamline generation and editing of iPSC for manifold applications.

This protocol describes in detail the generation of footprint-free induced pluripotent stem cells (iPSCs) from human pancreatic cells in feeder-free conditions, followed by editing using CRISPR/Cas9 ribonucleoproteins and characterization of the modified single-cell clones.

効率的な生成および CRISPR Cas9 システムを用いた人間の膵臓細胞から Ips をフィーダー フリーの編集

Embryonic and induced pluripotent stem cells can self-renew and differentiate into multiple cell types of the body. The pluripotent cells are thus coveted ...

Production of Genetically Engineered Golden Syrian Hamsters by Pronuclear Injection of the CRISPR/Cas9 Complex

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. The introduced genetic material may integrate into the embryonic genome and generate transgenic animals with foreign genetic information following transfer of the injected embryos to foster mothers. Following the success in mice, PN injection has been applied successfully in many other animal species. Recently, PN injection has been successfully employed to introduce reagents with gene-modifying activities, such as the CRISPR/Cas9 system, to achieve site-specific genetic modifications in several laboratory and farm animal species. In addition to mastering the special set of microinjection skills to produce genetically modified animals by PN injection, researchers must understand the reproduction physiology and behavior of the target species, because each species presents unique challenges. For example, golden Syrian hamster embryos have unique handling requirements in vitro such that PN injection techniques were not possible in this species until recent breakthroughs by our group. With our species-modified PN injection protocol, we have succeeded in producing several gene knockout (KO) and knockin (KI) hamsters, which have been used successfully to model human diseases. Here we describe the PN injection procedure for delivering the CRISPR/Cas9 complex to the zygotes of the hamster, the embryo handling conditions, embryo transfer procedures, and husbandry required to produce genetically modified hamsters.

Pronuclear (PN) injection of the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein-9 nuclease (CRISPR/Cas9) system is a highly efficient method for producing genetically engineered golden Syrian hamsters. Herein, we describe the detailed PN injection protocol for the production of gene knockout hamsters with the CRISPR/Cas9 system.

CRISPR/Cas9 複合体の前核注入による遺伝子組み換えゴールデン ハムスターの生産

The pronuclear (PN) injection technique was first established in mice to introduce foreign genetic materials into the pronuclei of one-cell stage embryos. ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

単一細胞のゲノムの配列のための超高スループット マイクロ流体プラットフォーム

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes and analysis of complex structural variants. Nanopore platforms perform single-molecule sequencing by directly measuring the current changes mediated by DNA passage through the pores and can generate hundreds of kilobase (kb) reads with minimal capital cost. This platform has been adopted by many researchers for a variety of applications. Achieving longer sequencing read lengths is the most critical factor to leverage the value of nanopore sequencing platforms. To generate ultra-long reads, special consideration is required to avoid DNA breakages and gain efficiency to generate productive sequencing templates. Here, we provide the detailed protocol of ultra-long DNA sequencing including high molecular weight (HMW) DNA extraction from fresh or frozen cells, library construction by mechanical shearing or transposase fragmentation, and sequencing on a nanopore device. From 20-25 &#181;g of HMW DNA, the method can achieve N50 read length of 50-70 kb with mechanical shearing and N50 of 90-100 kb read length with transposase mediated fragmentation. The protocol can be applied to DNA extracted from mammalian cells to perform whole genome sequencing for the detection of structural variants and genome assembly. Additional improvements on the DNA extraction and enzymatic reactions will further increase the read length and expand its utility.

Long-read sequences greatly facilitate the assembly of complex genomes and characterization of structural variation. We describe a method to generate ultra-long sequences by nanopore-based sequencing platforms. The approach adopts an optimized DNA extraction followed by modified library preparations to generate hundreds of kilobase reads with moderate coverage from human cells.

全 Genomic DNA 分析のための超長いリード シーケンス

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes ...

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has facilitated advanced genetic modification of Saccharomyces cerevisiae, a model organism and workhorse in industrial biotechnology. CRISPR-associated protein 12a (Cas12a), an RNA-guided endonuclease with features distinguishable from Cas9 is applied in this work, further extending the molecular toolbox for genome editing purposes. A benefit of the CRISPR/Cas12a system is that it can be used in multiplex genome editing with multiple guide RNAs expressed from a single transcriptional unit (single CRISPR RNA (crRNA) array). We present a protocol for multiplex integration of multiple heterologous genes into independent loci of the S. cerevisiae genome using the CRISPR/Cas12a system with multiple crRNAs expressed from a single crRNA array construct. The proposed method exploits the ability of S. cerevisiae to perform in vivo recombination of DNA fragments to assemble the single crRNA array into a plasmid that can be used for transformant selection, as well as the assembly of donor DNA sequences that integrate into the genome at intended positions. Cas12a is pre-expressed constitutively, facilitating cleavage of the S.&#160;cerevisiae genome at the intended positions upon expression of the single crRNA array. The protocol includes the design and construction of a single crRNA array and donor DNA expression cassettes, and exploits an integration approach making use of unique 50-bp DNA connectors sequences and separate integration flank DNA sequences, which simplifies experimental design through standardization and modularization and extends the range of applications. Finally, we demonstrate a straightforward technique for creating yeast pixel art with an acoustic liquid handler using differently colored carotenoid producing yeast strains that were constructed.

CRISPR/Cas12a Multiplex Genome Editing of Saccharomyces cerevisiae and the Creation of Yeast Pixel Art

The CRISPR/Cas12a system in combination with a single crRNA array enables efficient multiplex editing of the S.&#160;cerevisiae genome at multiple loci simultaneously. This is demonstrated by constructing carotenoid producing yeast strains which are subsequently used to create yeast pixel art.

CRISPR/Cas12a サッカロマイセスセレビシエの多重ゲノム編集と酵母ピクセルアートの創造

High efficiency, ease of use and versatility of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) ...

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various factors affect the relaxation rates, such as buffer composition, solution pH, temperature, and magnetic field. In this last regard, the spin-lattice relaxation time can be measured at clinical field strengths, but at lower fields, where these compounds are dispensed from the polarizer and transported to the MRI, the relaxation is even faster and difficult to measure. To have a better understanding of the amount of magnetization lost during transport, we used fast field-cycling relaxometry, with magnetic resonance detection of 13C nuclei at ~0.75 T, to measure the nuclear magnetic resonance dispersion of the spin-lattice relaxation time of hyperpolarized [1-13C]pyruvate. Dissolution dynamic nuclear polarization was used to produce hyperpolarized samples of pyruvate at a concentration of 80 mmol/L and physiological pH (~7.8). These solutions were rapidly transferred to a fast field-cycling relaxometer so that relaxation of the sample magnetization could be measured as a function of time using a calibrated small flip angle (3&#176;-5&#176;). To map the T1 dispersion of the C-1 of pyruvate, we recorded data for different relaxation fields ranging between 0.237 mT and 0.705 T. With this information, we determined an empirical equation to estimate the spin-lattice relaxation of the hyperpolarized substrate within the mentioned range of magnetic fields. These results can be used to predict the amount of magnetization lost during transport and to improve experimental designs to minimize signal loss.

Measuring the Spin-Lattice Relaxation Magnetic Field Dependence of Hyperpolarized [1-13C]pyruvate

We present a protocol to measure the magnetic field dependence of the spin-lattice relaxation time of 13C-enriched compounds, hyperpolarized by means of dynamic nuclear polarization, using fast field-cycled relaxometry. Specifically, we have demonstrated this with [1-13C]pyruvate, but the protocol could be extended to other hyperpolarized substrates.

過偏光のスピン格子緩和磁場依存性の測定[1-13C]ピルビン酸

The fundamental limit to in vivo imaging applications of hyperpolarized 13C-enriched compounds is their finite spin-lattice relaxation times. Various ...

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

The corn planthopper, Peregrinus maidis, is&#160;a pest of maize and a vector of several maize viruses. Previously published methods describe the triggering of RNA interference (RNAi) in P. maidis through microinjection of double-stranded RNAs (dsRNAs) into nymphs and adults. Despite the power of RNAi, phenotypes generated via this technique are transient and lack long-term Mendelian inheritance. Therefore, the P. maidis toolbox needs to be expanded to include functional genomic tools that would enable the production of stable mutant strains, opening the door for researchers to bring new control methods to bear on this economically important pest. However, unlike the dsRNAs used for RNAi, the components used in CRISPR/Cas9-based genome editing and germline transformation do not easily cross cell membranes. As a result, plasmid DNAs, RNAs, and/or proteins must be microinjected into embryos before the embryo cellularizes, making the timing of injection a critical factor for success. To that end, an agarose-based egg-lay method was developed to allow embryos to be harvested from P. maidis females at relatively short intervals. Herein are provided detailed protocols for collecting and microinjecting precellular P. maidis embryos with CRISPR components (Cas9 nuclease that has been complexed with guide RNAs), and results of Cas9-based gene knockout of a P. maidis eye-color gene, white, are presented. Although these protocols describe CRISPR/Cas9-genome editing in P. maidis, they can also be used for producing transgenic P. maidis via germline transformation by simply changing the composition of the injection solution.

Microinjection of Corn Planthopper, Peregrinus maidis, Embryos for CRISPR/Cas9 Genome Editing

Herein are protocols for collecting and microinjecting precellular corn planthopper embryos for the purpose of modifying their genome via CRISPR/Cas9-based genome editing or for the addition of marked transposable elements through germline transformation.

トウモロコシウンカ、 ペレグリヌスマイディス、CRISPR/Cas9ゲノム編集のための胚のマイクロインジェクション

The corn planthopper, Peregrinus maidis, is&#160;a pest of maize and a vector of several maize viruses. Previously published methods describe the ...

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the cell&#39;s survival requirements. Alternatively, carrying out DBTL cycles in cell-free environments can accelerate this process and alleviate concerns with host compatibility. A promising approach to cell-free metabolic engineering (CFME) leverages metabolically active crude cell extracts as platforms for biomanufacturing and for rapidly discovering and prototyping modified proteins and pathways. Realizing these capabilities and optimizing CFME performance requires methods to characterize the metabolome of lysate-based cell-free platforms. That is, analytical tools are necessary for monitoring improvements in targeted metabolite conversions and in elucidating alterations to metabolite flux when manipulating lysate metabolism. Here, metabolite analyses using high-performance liquid chromatography (HPLC) coupled with either optical or mass spectrometric detection were applied to characterize metabolite production and flux in E. coli S30 lysates. Specifically, this report describes the preparation of samples from CFME lysates for HPLC analyses using refractive index detection (RID) to quantify the generation of central metabolic intermediates and by-products in the conversion of low-cost substrates (i.e., glucose) to various high-value products. The analysis of metabolite conversion in CFME reactions fed with 13C-labeled glucose through reversed-phase liquid chromatography coupled to tandem mass spectrometry (MS/MS), a powerful tool for characterizing specific metabolite yields and lysate metabolic flux from starting materials, is also presented. Altogether, applying these analytical methods to CFME lysate metabolism enables the advancement of these systems as alternative platforms for executing faster or novel metabolic engineering tasks.

The protocols describe high-performance liquid chromatography methods coupled to refractive index or mass spectrometric detection for studying metabolic reactions in complex lysate-based cell-free systems.

リセートベースの無細胞システムにおける代謝物プロファイリングのためのリキッドクロマトグラフィー結合と屈折率または質量分析法検出

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the ...

マイクロインジェクションウエスタントウモロコシ Rootworm、 Diabrotica virgifera virgifera胚の生殖細胞への変換、または CRISPR/Cas9 ゲノム編集

要約

さらに動画を探す

この動画の章

のためのウイルス粒子

マイクロインジェクションアッセイをキズや

効率的な生成および CRISPR Cas9 システムを用いた人間の膵臓細胞から Ips をフィーダーフリーの編集

CRISPR/Cas9 複合体の前核注入による遺伝子組み換えゴールデンハムスターの生産

単一細胞のゲノムの配列のための超高スループットマイクロ流体プラットフォーム

全 Genomic DNA 分析のための超長いリードシーケンス

CRISPR/Cas12a サッカロマイセスセレビシエの多重ゲノム編集と酵母ピクセルアートの創造

過偏光のスピン格子緩和磁場依存性の^測定[1-13C]ピルビン酸

トウモロコシウンカ、ペレグリヌスマイディス、CRISPR/Cas9ゲノム編集のための胚のマイクロインジェクション

リセートベースの無細胞システムにおける代謝物プロファイリングのためのリキッドクロマトグラフィー結合と屈折率または質量分析法検出

マイクロインジェクション ウエスタン トウモロコシ Rootworm、 Diabrotica virgifera virgifera胚の生殖細胞への変換、または CRISPR/Cas9 ゲノム編集

要約

さらに動画を探す

この動画の章

のためのウイルス粒子

マイクロインジェクションアッセイをキズや

効率的な生成および CRISPR Cas9 システムを用いた人間の膵臓細胞から Ips をフィーダー フリーの編集

CRISPR/Cas9 複合体の前核注入による遺伝子組み換えゴールデン ハムスターの生産

単一細胞のゲノムの配列のための超高スループット マイクロ流体プラットフォーム

全 Genomic DNA 分析のための超長いリード シーケンス

CRISPR/Cas12a サッカロマイセスセレビシエの多重ゲノム編集と酵母ピクセルアートの創造

過偏光のスピン格子緩和磁場依存性の測定[1-13C]ピルビン酸

トウモロコシウンカ、 ペレグリヌスマイディス、CRISPR/Cas9ゲノム編集のための胚のマイクロインジェクション

リセートベースの無細胞システムにおける代謝物プロファイリングのためのリキッドクロマトグラフィー結合と屈折率または質量分析法検出

マイクロインジェクションウエスタントウモロコシ Rootworm、 Diabrotica virgifera virgifera胚の生殖細胞への変換、または CRISPR/Cas9 ゲノム編集

効率的な生成および CRISPR Cas9 システムを用いた人間の膵臓細胞から Ips をフィーダーフリーの編集

CRISPR/Cas9 複合体の前核注入による遺伝子組み換えゴールデンハムスターの生産

単一細胞のゲノムの配列のための超高スループットマイクロ流体プラットフォーム

全 Genomic DNA 分析のための超長いリードシーケンス

過偏光のスピン格子緩和磁場依存性の^測定[1-13C]ピルビン酸

トウモロコシウンカ、ペレグリヌスマイディス、CRISPR/Cas9ゲノム編集のための胚のマイクロインジェクション