私たちは、原稿上の有益なコメント代表的な結果を図3とジェイソンワットとクリス·ウェブスターのような予備実験を行うためのクリス·ウェブスターに感謝します。この研究のための資金はJLWに国立衛生研究所（米国）（R01DK074114）からの助成金によって提供された。この研究で使用されている一部の線虫株は研究基盤プログラムのNIHのオフィス（P40のOD010440）によって資金を供給される線虫遺伝学センターから提供された。

多価不飽和脂肪酸は、熱、光および酸素に敏感である。脂肪酸補充板を製造する場合、したがって、注意を払わなければならない脂肪酸が過剰な熱および光にさらされないようにする。 0.1％タージトール（NP-40）を含有するNGM培地は、脂肪酸ナトリウム塩を一定に攪拌しながら添加した後、オートクレーブし、部分的に冷却される。プレートを暗所で乾燥させる。 C.による脂肪酸の取り込みこれらのプレート上で培養エレガンスは、次いでガスクロマトグラフィーによって監視することができる。 1。脂肪酸補充培地の調製<ol><li>適切なサイズのフラスコに培地成分を測定します。 1 Lあたり、17グラムバクト寒天2.5グラムトリプトン3のNaCl、エタノールに溶解し1ミリリットル5 mg / mlのコレステロール、及び水10ミリリットルに溶解した10％タージトールを加える。 </li><li>ミリポア水の最終所望の​​体積の80％を追加して、別の数と同じ（空のガラスびんと一緒にメディアをオートクレーブ脂肪酸濃度が試験される）、ならびに適切なのメスシリンダー。 </li><li> 55℃に設定した水浴中でクールなメディア媒体は、ガラスバイアルをこじ開け安全注意事項を使用し、ガラス粒子は、脂肪酸で混合しない保証することにより、脂肪酸ナトリウム塩作業ストック溶液を調製し、冷却される。 100 mMのワーキングストックを作るために十分な脂肪酸を量る。 </li><li>精製水で100 mMの最終濃度までの脂肪酸溶液をもたらす。完全に脂肪酸ナトリウム塩を溶解することは、典型的には約20〜30分を要する。不活性ガスとの接触は酸化を防ぐので、アルゴン又は窒素を有する脂肪酸の​​作業ストックをパージする。バイアルに蓋をし、メディアが冷却されるまで暗闇での作業の在庫を保管してください。 </li><li> （任意）メディアのRNAiが所望される場合、ここにアンピシリンおよびイソプロピルβ-D-1-チオガラクトピラノシド（IPTG）の溶液を測定する。 </li><li>寒天は55℃に冷却したときは、1Lあたりの追加C：11 MのMgSO 4 mlの1 M CaCl 2を 1mlのリン酸緩衝液25mlの（1 Lあたり108.3グラムのKH 2 PO 4、35.6グラムのK 2 HPO 4は、オートクレーブ処理）。 RNAiのプレートを作った場合、アンピシリンおよびIPTGソリューション滅菌フィルタを追加します。プレートのすべてのタイプのために、1 Lに、最終的なメディアボリュームを持って来るために滅菌水を追加</li><li>火気の近くで、オートクレーブ処理にメディアを転送し、適切にメスシリンダーをサイズにし、目的のボリュームに暖かい滅菌水を加えます。戻って最初のフラスコにメディアを転送し、攪拌して混合します。 </li><li>メスシリンダーにオートクレーブ処理し、適切な大きさで測定して、試験される濃度の数に等しい数のボトルへの火炎、転写媒体近。脂肪酸作業ストックが完全に溶解するまで水浴を使用して液体として等分メディアを維持する。 </li><li>メディアが触ると暖かく、しかし熱くないまで撹拌プレートおよび撹拌に1瓶を置きます。十分な自分を残していることを確認してください脂肪酸原液を加えて混ぜるとメディアが固化し始める前に、プレートを注ぐための時間。撹拌プレートは、温度制御されている場合は、55℃に設定し</li><li>所望の最終濃度のために培地に100mMの脂肪酸作業ストックに希釈する。白色の沈殿が溶液（約1分）になるまで、メディアを攪拌する。メディアはまだ、その後、わずかに白濁することがあります。 </li><li> RNAiのメディアが必要な場合（オプション）2 mMの最終濃度に添加し、IPTGを0.1mg /アンピシリンを追加します。 </li><li>培地を60mmプレート又は媒体100ミリメートル当たりプレート当たり25mlの培地を8mlを添加し、自動化ピペットエイドおよび滅菌を25mlピペットを使用して注ぐ。残りのボトル用のステップを注ぐ脂肪酸に加え、プレートを繰り返します。 </li><li>寒天が固化した後、光酸化から守るために、よく通気引き出しの中に、室温でボックスまたはストアと脂肪酸補充プレートをカバーしています。 </li><li>シードE.プレートが乾燥してきた2日後にプレートに大腸菌 OP50。60ミリメートルプレート、ピペット（37℃で増殖）一晩OP50文化300μlのため。 RNAiのプレートを注入した場合には、プレートを4日間乾燥した後に、RNAi細菌300μlのシード。プレートの乾燥時間は、様々な実験室環境における湿度の差に調整される必要があり得る。 </li><li>細菌ローンが乾燥している間、室温で暗い環境での培養する。 </li></ol> 2。 DGLAの補充によって生殖細胞破壊を誘発する<ol><li> C.が0.3mm DGLA（午後8時03分、N-6）を含むプレート上で成長した虫は、両方の幼虫期と成人線虫における生殖細胞の破壊に無菌になる。 <ol><li>アルカリ性次亜塩素酸溶液と妊娠雌雄同体を処理することにより、L1幼虫の同期化された人口を準備（10ミリリットルソリューションの：5MのNaOH、家庭用漂白剤を2.5ミリリットル、および7ミリリットルのH 2 Oを0.5ミリリットル）。優しく成虫まで、この溶液中の妊娠雌雄同体を揺するSが溶解する。卵は保持され、低速遠心分離によってペレット化することができる。 </li><li> （3グラムのKH 2 PO 4、6グラムHPO 4、5グラムのNaCl、1ミリリットルの1MのMgSO 4、H 2 O Lでオートクレーブ後のMgSO 4を追加する1〜2 Na）のM9バッファーに卵の準備を3回洗浄します。十分な酸素を提供するために、一晩振とう機上でM9バッファーやロックの5ミリリットルの最終容量に15ミリリットルのプラスチックチューブ内に卵を懸濁します。 </li><li>のL1幼虫は2日OP50、またはRNAi細菌をめっき後1日で播種後補足プレートに添加する必要があります。彼らは大人の段階に到達するまで3日間、20℃でワームをインキュベートか。 </li></ol></li><li>成虫は、子宮内発育する胚を可視化するために十分に高い倍率で解剖顕微鏡を使用して無菌性についてスコア化することができます。成功した生殖細胞の損失は、卵の明確な子宮無効として表示されます。 </li><li>ワームはまた、核酸aで固定し、染色によりスコア化することができますCID染料diamindinophenylindole（DAPI）、生殖細胞および発生中の胚中の核の可視化を容易にする。迅速なDAPI染色は時計皿9にM9バッファーの低下にワームを選ぶことによって達成される。 <ol><li> 0.2 ngの/ mで1ミリリットルと洪水ウォッチガラス。 DAPI 95％エタノール溶液中で、約5分間放置します。 </li><li>スライド上のベクタシールドのドロップにワームを選び、その後、カバーガラスでカバーしています。 </li><li>最適な染色のため、暗い一晩中4℃で密封カバーガラスとストアは、しかし、スライドは直ちに表示することができます。 UVランプとフィルターを備えた蛍光顕微鏡を用いて生殖細胞核の存在または非存在についてスコア。 </li></ol></li></ol> 3。ガスクロマトグラフィーによる脂肪酸摂取を確認する C.全体の脂肪酸組成線虫を分離し、ガスのchrを使用して定量化される脂肪酸メチルエステル（FAME）を製造することによって決定することができるomatography 4。 <ol><li>水でプレートからそれらを洗浄し、シラン化された13ミリメートルX 100ミリメートルガラススクリュートップチューブに移すことによって、500〜1,000成虫を集める。 </li><li>ワームは、重力によって沈降してみましょう、その後、ガラスパスツールピペットを用いて、できるだけ多くの水を除去する。 </li><li>水で一度ワームを洗ってから、再度、できるだけ多くの水を除去する。 </li><li> FAMEを、水浴中で1時間、70℃で1〜2.5％のH 2 mlのメタノール中のSO 4、次いで加熱を加えることによって形成されている。 </li><li>水浴からチューブを外し、1分間冷やす。 </li><li> 1.5ミリリットルの水とヘキサンの0.25ミリリットルを追加することにより、脂肪酸メチルエステルを抽出します。 </li><li>要約チューブ、激しく振る。 </li><li>水系溶媒からヘキサンを分離するために最高速度で1分間卓上臨床遠心分離機中で遠心管。 </li><li> GCバイアルにヘキサン（最上層）を転送する水相のいずれかを転送しないように注意して、GC用バイアル内に挿入します。 GCのAのnalysis、ヘキサン中のFAMEの1-2μlのFAMEの分析に適した極性のキャピラリーガスクロマトグラフィーカラムに注入する。アジレント7890 GCインジェクタ1.4 ml /分の流速で、250℃に設定し、GCオーブン内で1分間保持され、130℃の初期温度にプログラムされている。その後、温度を190℃まで10℃/分の傾斜した後、210℃まで5℃/分で再び上昇させ、さらに1分間保持した。 </li><li> DGLAの取り込みが発生している保証するために、C.の同定のための真正標準物質を使用し、水素炎イオン化検出器（FID）または質量分析（MS）検出によって脂肪酸メチルエステルの分析脂肪酸のエレガンス 。 </li></ol>

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著者は、彼らが競合する経済的利益を持っていないことを宣言します。

ここでは、C.の補充方法を記載食事性の不飽和脂肪酸との虫 。前述したようなPUFAの二重結合の反応性の性質はこれらの脂肪酸は、熱と光11によって酸化に敏感であることが原因となるので、お手入れは、PUFAを補足プレートの準備に注意する必要があります。酸化を避けるために、メディアが暗い環境下で55℃を記憶プレートに冷却した後に液体寒天培地にPUFAを添加する?...

脂肪酸は膜の必須構造成分、ならびに効率的なエネルギー貯蔵分子である。さらに、脂肪酸は、リパーゼにより細胞膜から切断することができ、酵素的にシグナル伝達エフェクター1を産生するように改変することができる。天然に存在する多価不飽和脂肪酸（PUFA）は、二つまたはそれ以上のシス二重結合を含有する。オメガ-3脂肪酸とオメガ-6脂肪酸は、脂肪酸のメチル末端に対する二重結合の位置に基づいて互いに区別される。健康な食事は、ω-6およびω-3脂肪酸の両方を必要とする。しかし、西洋の食事はオメガ-6脂肪酸とオメガ-3脂肪酸に劣るが特に豊富である。高オメガ6オメガ-3脂肪酸比は、心血管疾患および炎症性疾患のリスクの増加、しかし、特定の脂肪酸の正確な有益と有害な機能はウェル2に理解されていないと関連している。回虫線虫elegaそれはω-3デサチュラーゼ、ほとんどの動物3,4には存在しない活性を含むオメガ-6およびオメガ-3脂肪酸の範囲を生成するために必要な酵素の全てを合成するため、ナノ秒は、脂肪酸の機能を研究するのに有用である。脂肪酸不飽和化酵素を欠いた変異体が発達し、神経学的欠陥4-6の範囲につながる、特定のPUFAを生産することができない。 食事性脂肪酸の生理的効果を研究するために、我々はCの両方の変異体およびRNAiノックダウン技術を使用して、遺伝子解析と互換性の生化学的ア ​​ッセイを開発してきた虫 。特定のPUFA補充の前注ぐ寒天培地への脂肪酸のナトリウム塩溶液を添加することによって達成される。これはEで、PUFA摂取になりそれは細菌膜に蓄積大腸菌食料源、。C.虫は、PUFAを含む細菌を摂取し、この栄養補給は、欠陥品を救出するのに十分であるPUFA欠損変異体のTS。ほとんどの脂肪酸の補給は、野生型動物に有害な影響が、特定のオメガ-6脂肪酸、特に、ジホモ-γ-リノレン酸（DGLA、20:3のn-6）C.の永久的な破壊を引き起こすことがありませんエレガンス生殖細胞7,8。 ガスクロマトグラフィーは、細菌の食料源（OP50又はHT115のいずれか）ならびに線虫に補充脂肪酸の取り込みを監視するために使用される。培地中の界面活性剤タージトール（NP-40）の添加は、全体のプレートとE.による脂肪酸のより効率的な取り込みを介して脂肪酸の均一な分布を可能にする大腸菌や線虫。我々は、不飽和脂肪酸が容易に細菌およびC.によって取り込まれることを見出した虫が、飽和脂肪酸の取り込みはあまり効率的である。この記事では、脂肪酸と寒天培地を補足する方法、ならびに第における脂肪酸の​​取り込みを監視する方法を段階的に説明するガスクロマトグラフィーを使用して電子線虫。

<table border="0" cellpadding="0" cellspacing="0" width="765">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Bacto-Agar</td>
			<td style="width:128px;">Difco</td>
			<td style="width:155px;">214010</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Tryptone</td>
			<td style="width:128px;">Difco</td>
			<td style="width:155px;">211705</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">NaCl</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3624-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Tergitol</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">NP40S-500mL</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Cholesterol</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">C8667-25G</td>
			<td style="width:275px;">(5 mg/mL in ethanol)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">MgSO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">2504-01</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">CaCl2</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">1311-01</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">K2HPO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3254-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">KH2PO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">3246-05</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sodium dihomogamma linolenate</td>
			<td style="width:128px;">NuCHEK</td>
			<td style="width:155px;">S-1143</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Warm sterile Millipore water</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sterile water for collecting worms</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Nuclease-free Water for DGLA stock solution</td>
			<td style="width:128px;">Ambion</td>
			<td style="width:155px;">AM9932</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Ampicillin</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">BP1760-25</td>
			<td style="width:275px;">100 mg/ml in water (for RNAi plates)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Isopropyl-beta-D-thiogalactopyranoside (IPTG)</td>
			<td style="width:128px;">Gold Biotechnology</td>
			<td style="width:155px;">12481C100</td>
			<td style="width:275px;">1 M in water (for RNAi plates)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">HSO4</td>
			<td style="width:128px;">J.T. Baker</td>
			<td style="width:155px;">9681-03</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Methanol</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">A452-4</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Hexane</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:155px;">H302-4</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">diamindinophenylindole (DAPI)</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:155px;">D9542</td>
			<td style="width:275px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">VectaShield</td>
			<td style="width:128px;">Vector Laboratories</td>
			<td style="width:155px;">H-1000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Glass Flask</td>
			<td style="width:128px;">Corning</td>
			<td style="width:155px;">4980-2L</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Autoclaveable Glass bottles with stirbars</td>
			<td style="width:128px;">Fisherbrand</td>
			<td style="width:155px;">FB-800</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Autoclaveable Glass Graduated Cylinder</td>
			<td style="width:128px;">Fisherbrand</td>
			<td style="width:155px;">08-557</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Stir Plate</td>
			<td style="width:128px;">VWR</td>
			<td style="width:155px;">97042-642</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Waterbath at 55+ &#176;C</td>
			<td style="width:128px;">Precision Scientific Inc.</td>
			<td style="width:155px;">66551</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Screwcap Brown Glass Vial</td>
			<td style="width:128px;">Sun SRI</td>
			<td style="width:155px;">200 494</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Argon gas tank</td>
			<td style="width:128px;"></td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Automated Pipette aid</td>
			<td style="width:128px;">Pipette-Aid</td>
			<td style="width:155px;">P-90297</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:208px;">Sterile Serological Pipettes (25 ml)</td>
			<td style="width:128px;">Corning</td>
			<td style="width:155px;">4489</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Bunsen Burner</td>
			<td style="width:128px;">VWR</td>
			<td style="width:155px;">89038-534</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Dissection microscope</td>
			<td style="width:128px;">Leica</td>
			<td style="width:155px;">TLB3000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Silanized glass tube</td>
			<td style="width:128px;">Thermo Scientific</td>
			<td style="width:155px;">STT-13100-S</td>
			<td>for FAMEs derivitization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:208px;">PTFE Screw caps</td>
			<td style="width:128px;">Kimble-Chase</td>
			<td style="width:155px;">1493015D</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Clinical tabletop centrifuge</td>
			<td style="width:128px;">IEC</td>
			<td style="width:155px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Crimp Vial</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Vial Insert</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 232</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">GC Vial cap</td>
			<td style="width:128px;">SUN SRi</td>
			<td style="width:155px;">200 100</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Gas Chromatograph</td>
			<td style="width:128px;">Agilent</td>
			<td style="width:155px;">7890A</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Mass Spectrometry Detector</td>
			<td style="width:128px;">Agilent</td>
			<td style="width:155px;">5975C</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:208px;">Column for gas chromatography</td>
			<td style="width:128px;">Suppelco</td>
			<td style="width:155px;">SP 2380</td>
			<td>30 m x 0.25 mm fused silica capillary column</td>
		</tr>
	</tbody>
</table>

dietary supplementation of polyunsaturated fatty acids in caenorhabditis elegans

脂肪酸は、非常に多くの細胞機能に必須である。それらは、膜の疎水性コアを構成し、効率的なエネルギー貯蔵分子を提供し、種々のシグナル伝達経路に関与する。 線虫（Caenorhabditis elegans）は、オメガ-6及びオメガ-3脂肪酸の範囲を生成するのに必要な酵素の全てを合成する。これは、簡単な解剖学および利用可能な遺伝的手段の範囲と組み合わせると、脂肪酸の機能を研究するための魅力的なモデルにする。食餌性脂肪酸の生理学的効果を媒介する遺伝的経路を調べるために、我々はC.を補足する方法を開発した不飽和脂肪酸とエレガンスダイエット。補充は、ワームの脂肪酸組成を変化させる有効な手段であり、また、脂肪酸欠損変異体における欠損をレスキューするために使用することができる。我々の方法は、脂肪acidsodium塩を補充した線虫の増殖培地寒天（NGM）を使用しています。 incorporaなっ補足プレート中の脂肪酸次いで、C.によって取り込まれる細菌の食物源の膜へテッド補足細菌を餌虫 。また、補充ワームで発生する脂肪酸組成の変化を監視するために、ガスクロマトグラフィープロトコルを記載している。これはCの大小の集団の食生活を補完する効率的な方法です虫 、このメソッドの応用範囲を可能にする。

Cの補充食虫 、細菌膜への脂肪酸取り込みおよび組み込む細菌の食物源の能力によって制限される。 Eの能力を決定するその膜内に様々な脂肪酸資化大腸菌 OP50は、OP50はノーサプリメント、0.1 mMおよびステアリン酸（午前18時00分）、オレイン酸ナトリウム（18:1のn-9）およびナトリウムDGLA（20 0.3mMの濃度で培地上にプレーティングした：3nは-6）。プレートを暗所で2?...

Watch this Scientific Journal Video about Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans at JoVE.com

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

の&lt;em&gt;線虫のエレガンは&lt;/ em&gt;は、開発と生理学で多価不飽和脂肪酸の機能を探求するための有用なモデルである。このプロトコルは、の&lt;em&gt; Cを補充する効率的な方法を説明します虫&lt;/ em&gt;の多価不飽和脂肪酸とダイエット。

多価不飽和脂肪酸の食事補給<em&gt;線虫（Caenorhabditis elegans）</em

Fatty acids are essential for numerous cellular functions. They serve as efficient energy storage molecules, make up the hydrophobic core of membranes, ...

dietary-supplementation-polyunsaturated-fatty-acids-caenorhabditis

Research

JoVE Journal

Biology

Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

17.0K ビュー.  Washington State University. の線虫のエレガンはは、開発と生理学で多価不飽和脂肪酸の機能を探求するための有用なモデルである。このプロトコルは、の Cを補充する効率的な方法を説明します虫の多価不飽和脂肪酸とダイエット。 

ビデオ: Dietary Supplementation of Polyunsaturated Fatty Acids in Caenorhabditis elegans

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

アイソレーションと試験管内で</emの>活性化線虫（Caenorhabditis elegans）精子

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

生物学

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide range of species, including mammals. It also causes a decrease in body weight and fertility, as well as lower levels of plasma glucose, insulin, and IGF-1 in these animals. This treatment is often referred to as dietary restriction (DR) or caloric restriction (CR). The nematode Caenorhabditis elegans has emerged as an important model organism for studying the biology of aging. Both environmental and genetic manipulations have been used to model DR and have shown to extend lifespan in C. elegans. However, many of the reported DR studies in C. elegans were done by propagating animals in liquid media, while most of the genetic studies in the aging field were done on the standard solid agar in petri plates. Here we present a DR protocol using standard solid NGM agar-based plate with killed bacteria.

Solid Plate-based Dietary Restriction in Caenorhabditis elegans

Here we present a protocol for performing solid plate-based dietary restriction in C. elegans with killed bacteria.

固体プレートベースの食餌制限線虫（Caenorhabditis elegans）</em

Reduction of food intake without malnutrition or starvation is known to increase lifespan and delay the onset of various age-related diseases in a wide ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

の初期発生のタイムラプス顕微鏡線虫（Caenorhabditis elegans）

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

基本的な<em&gt;線虫Caenorhabditis elegans</em&gt;方法：同期との観測

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

におけるプロスタグランジンの抽出と分析<em&gt;線虫（Caenorhabditis elegans）</em

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we know about C. elegans is limited to laboratory cultivation of the nematodes that may not necessarily reflect the environments they normally inhabit in nature. Cultivation of C. elegans in a 3D habitat that is more similar to the 3D matrix that worms encounter in rotten fruits and vegetative compost in nature could reveal novel phenotypes and behaviors not observed in 2D. In addition, experiments in 3D can address how phenotypes we observe in 2D are relevant for the worm in nature. Here, a new method in which C. elegans grows and reproduces normally in three dimensions is presented. Cultivation of C. elegans in Nematode Growth Tube-3D (NGT-3D) can allow us to measure the reproductive fitness of C. elegans strains or different conditions in a 3D environment. We also present a novel method, termed Nematode Growth Bottle-3D (NGB-3D), to cultivate C. elegans in 3D for microscopic analysis. These methods allow scientists to study C. elegans biology in conditions that are more reflective of the environments they encounter in nature. These can help us to understand the overlying evolutionary relevance of the physiology and behavior of C. elegans we observe in the laboratory.

Cultivation of Caenorhabditis elegans in Three Dimensions in the Laboratory

We present a simple method to construct 3D nematode cultivation systems called NGT-3D and NGB-3D. These can be used to study nematode fitness and behaviors in habitats that are more similar to natural Caenorhabditis elegans habitats than the standard 2D laboratory C. elegans culture plates.

の栽培<em&gt;線虫（Caenorhabditis elegans）</em&gt;ラボにおける三次元で

The use of genetic model organisms such as Caenorhabditis elegans has led to seminal discoveries in biology over the last five decades. Most of what we ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

そのまま線虫の酸素消費量の測定

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

細胞外小胞体のカエノールハブディティス・エレガンスの精製と分析

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

カエノハブディティス・エレガンスにおける組織特異的プロテオスタティック低下の定量化

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

Caenorhabditis elegansにおける凝集定量の自動化

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...