我々はすべてコールドウェル研究室のメンバーの協力精神を認識したい。ラボにおける運動障害の研究は、バッハマン-シュトラウスジストニア＆パーキンソン財団、米国パーキンソン財団、米国パーキンソン病協会、アラバマ州のパーキンソン病協会、パーキンソン病研究のためのマイケルJフォックス財団、および学部研究によってサポートされています

A.発現プラスミドの構築二つのプラスミドが必要です。興味と選択可能な形質転換マーカー（プラスミドマーカーは、研究コミュニティ内から通常使用可能ですが）などの第二の遺伝子の組織特異的発現のための1つを。 実験的なプラスミド<ol><li>興味の組織/細胞型において発現されるプロモーターを選択し、この場合は、DAトランスポーター（Pdat - 1）プロモーターが使用されます。この発現プラスミドは、組換えクローニングを1プロモーターの関心の下流の任意の遺伝子の挿入を可能にするインビトロジェンのゲートウェイシステムと互換性のあるデスティネーションベクター（PDEST - DAT - 1）として作成されました。 </li><li>目的の遺伝子のcDNAをPCRには、ゲートウェイATT B組換え配列を含むプライマーを用いて増幅される。増幅したcDNAは、最初のエントリのベクトルを作成するためにドナーベクターpDONR201に再結合し、大腸菌株DH5αに変換されます。成功した組換え体とDNAのミニプレップ分離の選択後、エントリーベクターは、発現プラスミドを作成するPDEST - DAT - 1デスティネーションベクターに再結合されています。組み換えクローン作成の詳細については、InvitrogenのGatewayのマニュアルから、またはコールドウェルらで用意されています。5。 </li></ol>プラスミド選択可能なマーカープラスミド形質転換マーカーは明らか組織における蛍光タンパク質の発現を駆動するプロモーターを持つベクトルで構成されています。この特定の手順では、UNC - 54プロモーターは、体壁筋の桜のタンパク質発現（：：チェリーPunc - 54）を駆動します。 マイクロインジェクションを介してトランスジェニック線虫のB.の生成 ：関連Joveの記事を参照してください。 <a href="https://www.jove.com/index/details.stp?ID=833">https://www.jove.com/index/details.stp?ID=833を</a> DAの神経変性の解析のためのC.遺伝子交雑<ol><li>ワームは、標準的な手順6を使用して栽培されています。 </li><li>プレイス10 Pdat - 1：：α- SYN、Pdat - 1：：（：：遺伝子XとPunc - 54：：チェリーPdat - 1を発現する）3-4 L4ステージトランスジェニック雌雄同体と交配プレート上にGFPの雄1、2 。これは、作成した3つの別々の安定した線のそれぞれに対して個別に実行する必要があります。 2日後、男性を取り外します。 </li><li> F1世代を点検し、いくつかの男性の子孫が存在する場合、交配が成功しました。 </li><li>それぞれのクロスから5両性L4動物をクローニングすること展示の両方Pdat - 1：：GFP蛍光マーカー（男性の親から継承されます）とPunc - 54：：チェリー蛍光体壁筋のマーカー（両性の親から継承されます）。クローン動物は、彼らはまだプレート上に存在する雄動物と交配されていないことを確認するためにL4段階にあるとする必要があります。それらが自己交差してF2子孫を生成することができます。 </li><li>ステップ3で生成したF2動物が出クローン化されています。具体的には、転送〜自分の個々のプレートに、両方の蛍光マーカーを示す50〜10の動物。画面DAニューロンの動物の100％がGFPを発現するプレートのF3世代や動物の一部が体壁筋細胞で発現する桜です。これらの動物はPdat - 1のホモ接合になります：：α- SYN、Pdat - 1：：GFP遺伝子を安定新しく作成された遺伝子（遺伝子X）を送信中。 </li></ol> D.ドーパミン作動性ニューロンの解析<ol><li>新鮮な上記で作成した3行のそれぞれのプレートだけでなく、Pdat - 1を準備します：：α- SYN、Pdat - 1：：GFP単独系統。彼らが大人に成長することができます。 </li><li>新鮮なプレート上にそれぞれのラインから30〜40トランスジェニック大人を転送する。それらを20℃で4時間胚を築くために許可する</li><li>大人を削除します。彼らはL4段階に到達するまでの胚は20℃で3〜4日間、開発することができます。 </li><li> 0.04 mg / mlの5 - フルオロ-2&#39; - デオキシウリジン（FUDR）を含むプレートに〜100トランスジェニックL4動物を選択してください。したがって、実験動物7を圧倒から子孫を防ぐDNA合成の阻害を介して次の世代のこのヌクレオチドアナログブロックの開発、。それは光に敏感であるため、光からFUDR板を保護する。 </li><li>成体動物は、産卵に続く様々な日で分析されます。分析の適切な日々は経験的に決定されています。例えば、我々は決定したその77％、87％、Pdat - 1の90％：：α- SYN、Pdat - 1：：GFPの動物はそれぞれ、日6、7および10後の開発で縮退DAニューロンを示す。したがって、我々が関心の導入遺伝子は神経変性を高める可能性があることを予測した場合、我々は6日目及び/または7で分析を行います。同様に、神経変性を抑制する可能性導入遺伝子は、7日目および10で分析されます。 </li><li> 4新鮮なアガロースパッドを（マイクロインジェクション寒天パッドとは異なり、これらの新鮮な使用されていると乾きが許可されていない）ことを確認します。それらの間でテープの部分を持つ2つの顕微鏡スライドを設定し、テープには、等価的に厚いパッドのためのスペーサーとして機能します。三分の一をレイベンチでそれらの間にスライドさせて（それらは三遅れないように配置されています）。中央のスライド上に溶融アガロースの低下を置き、すぐに垂直に3つのすべてのスライドにし、全体で、アガロースの上に別のスライドを置く。パッドの使用準備が整うまで、一緒に2つのスライドを残して、いくつかの追加のパッドを作る。 </li><li>ワームDAニューロンを分析するために、22 × 30mmのカバーガラス上の3 mMのレバミゾール（麻酔）の6μlのドロップを置きます。アガロースのパッド上にカバーガラスを反転して、ドロップに（10が30を超えて余分な得点する）40大人の動物を選んでください。他の行のそれぞれに対して繰り返します。 </li><li>落射蛍光及びFITCやGFPの可視化を可能にするフィルタキューブを持つ化合物の顕微鏡を使用して各CEPとADEニューロンの存在のためのスコアラインあたり30匹を。我々は、GFP HYQフィルターキューブ（クロマテクノロジー）を与える使用してください。添付のスコアリングシート上のデータを記録する。野生型の動物は、すべての4 CEPと2 ADEニューロンにおける完全なGFPの蛍光を保持します。 DAニューロンの喪失を示す個々の動物は、非WTまたは&quot;退化&quot;と記録されています。同様に、変性の程度は、各動物に失わ合計ニューロンと同様に人口を数えることによって得点することができます。 </li><li>分析を2回以上（試験ごとにラインごとに30以上の動物）を繰り返します。分析の三回から、野生型DAニューロンを示すワームの合計数が平均化されます。神経保護のための統計分析は、コントロールのワーム（：：α- SYN、Pdat - 1：Pdat - 1：GFP）を比較するために、学生のt -検定（P &lt;0.05）を用いて行われる株とそのDAニューロンで過剰発現する候補遺伝子。 </li></ol>

<ol>
	<li>Cao, S., Gelwix, C. C., Caldwell, K. A., Caldwell, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Torsin-mediated+neuroprotection+from+cellular+stresses+to+dopaminergic+neurons+of+C.+elegans.">Torsin-mediated neuroprotection from cellular stresses to dopaminergic neurons of C. elegans.</a> J Neurosci. 25, 3801-3812 (2005).</li><li>Hamamichi, S., Rivas, R. N., Knight, A. L., Cao, S., Caldwell, K. A., Caldwell, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypothesis-based+RNAi+screening+identifies+neuroprotective+genes+in+a+Parkinson&#x2019;s+disease+model.">Hypothesis-based RNAi screening identifies neuroprotective genes in a Parkinson&#x2019;s disease model.</a> PNAS. 105, 728-733 (2008).</li><li>Cooper, A. A., Gitler, A. D., Cashikar, A., Haynes, C. M., Hill, K. J., Bhullar, B., Liu, K., Xu, K., Strathearn, K. E., Liu, F., Cao, S., Caldwell, K. A., Caldwell, G. A., Marsischky, G., Kolodner, R. D., Labaer, J., Rochet, J. C., Bonini, N. M., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=alpha-synuclein+blocks+ER-golgi+traffic+and+Rab1+rescues+neuron+loss+in+Parkinson's+models.">alpha-synuclein blocks ER-golgi traffic and Rab1 rescues neuron loss in Parkinson's models.</a> Science. 313, 324-328 (2006).</li><li>Gitler, A. D., Bevis, B. J., Shorter, J., Strathearn, K. E., Hamamichi, S., Su, L. J., Caldwell, K. A., Caldwell, G. A., Rochet, J. -. C., McCaffery, J. M., Barlowe, C., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Parkinson's+disease+protein+alpha-synuclein+disrupts+cellular+Rab+homeostasis.">The Parkinson's disease protein alpha-synuclein disrupts cellular Rab homeostasis.</a> PNAS. 105, 145-150 (2008).</li><li>Caldwell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+Genomics:+A+Discovery-Based+Laboratory+Course.">Integrated Genomics: A Discovery-Based Laboratory Course.</a> , (2006).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77, 71-94 (1974).</li><li>Gandhi, S., Santelli, J., Mitchell, J. D. H., Stiles, J. W., Sanadi, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+maintaining+large,+aging+populations+of+Caenorhabditis+elegans.">A simple method for maintaining large, aging populations of Caenorhabditis elegans.</a> Mechanisms of Ageing and Development. 12, 137-150 (1980).</li></ol>

ドーパミンニューロンの年齢に依存する損失は、パーキンソン病の臨床的特徴であり、α-シヌクレインと呼ばれる蛋白質の蓄積や折り畳みに関連付けられている。ここでは、Cのドーパミン作動性ニューロン、蛍光遺伝子を経由して、ラベルの作成 ​​方法を示しています虫とこれらの細胞で共発現ヒトα-シヌクレインによるパーキンソン病に見られる神経変性を模倣する。 このビデオで...

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<td>Agarose</td>
<td>Ultrapure</td>
<td>Invitrogen</td>
<td>15510-027</td>
<td>&nbsp;</td>
<tr>
<td>Coverglass 20x30 mm</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-548-5A</td>
<td>&nbsp;</td>
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<td>Microscope Slides</td>
<td>Plain, 3x1"</td>
<td>Fisher</td>
<td>12-549</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting with Fluorescence</td>
<td>Microscope</td>
<td>Nikon</td>
<td>SMZ800</td>
<td>&nbsp;</td>
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<td>Dissecting</td>
<td>Microscope</td>
<td>Nikon</td>
<td>SMZ645</td>
<td>&nbsp;</td>
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<td>Epifluorescent</td>
<td>Microscope</td>
<td>Nikon</td>
<td>Model E-800</td>
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<td>Filter Cube, GFP HYQ</td>
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application of a c. elegans dopamine neuron degeneration assay for the validation of potential parkinson's disease genes

パーキンソン病（PD）の診断と治療の改善が危険にさらされる人口をレンダリング感受性要因に関する知識に依存しています。 PDに関連する新たな遺伝的要因を識別しようとする過程で、科学者は候補遺伝子、多型、および重要な進歩を表すタンパク質の多くのリストを生成しているが、これらのリード線は、機構的に未定義のまま。私たちの仕事は大幅に単純な動物モデルシステムの利点を活用することによってこのようなリストを狭めることに狙いを定めています。人間は神経細胞の十億を有しているが、顕微鏡回虫線虫Caenorhabditis elegansは、正確に302 hemaphroditesでのわずか8農産物のドーパミン（DA）があります。投与量と年齢に依存する神経変性におけるC. elegansのDAニューロンの結果でPD -関連タンパク質、α-シヌクレインをコードするヒト遺伝子の発現。 DAニューロンのヒトα-シヌクレインを発現しているワームは同質であり、DAトランスポーターのプロモーター下にGFPとα-シヌクレインのヒト（Pdat - 1）の両方を表現する。 GFPの存在は、これらの動物ではDA神経変性を以下のための容易に視覚化マーカーとして機能します。我々は最初にα-シヌクレイン誘発性のDA神経変性がtorsinA、分子シャペロン活性が1の蛋白質によってこれらの動物で救助できることを実証した。さらに、候補PD関連遺伝子は、C. elegansのDAニューロンで過剰発現されたα-シヌクレインのミスフォールディングのアッセイを用いて大規模なRNAiスクリーニングの取り組みを経由して私たちの研究室で同定した。我々は、彼らがα-シヌクレイン誘発性のDA神経変性2を救出したとして7遺伝子の5人がPDの表現の重要な候補モジュレーターをテストしたことが判明。また、リンドクイストラボ（Joveのこの問題は）酵母の画面を実行したことにより、α-シヌクレイン依存毒性は細胞毒性を増強または抑制することができる遺伝子の読み出しとして使用されます。私たちは、その後、私たちの線虫α-シヌクレイン誘発神経変性のアッセイで酵母の候補遺伝子を調べ、正常にこれらの目標3、4の多くの検証。 私たちの方法論はPdat - 1プロモーターの制御下に候補遺伝子のcDNAの組換えクローニングを使用して、C. elegansのDAニューロン特異的発現ベクターの生成を伴います。これらのプラスミドは、成功した変換のための選択マーカーと共に、野生型（N2）ワームにマイクロインジェクションされています。 DAニューロンの候補タンパク質を生産する複数の安定したトランスジェニック系統が得られ、その後独立してα-シヌクレインの変性株に交差させ、老化の過程で、動物と個々のニューロンレベルの両方で、神経変性について評価されています。

Watch this Scientific Journal Video about Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes at JoVE.com

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson&#039;s Disease Genes

このビデオでは、パーキンソン病のモデルとしてドーパミン作動性ニューロンの神経変性を評価するために線虫を使用する方法を示します。さらに、遺伝子スクリーニングは、どちら変性を高めたり、神経保護の要因を識別するために使用されます。

潜在的なパーキンソン病の遺伝子を検証するための線虫のドーパミンニューロンの変性のアッセイのアプリケーション

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

application-c-elegans-dopamine-neuron-degeneration-assay-for

Research

JoVE Journal

Biology

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

15.9K ビュー.  University of Alabama. このビデオでは、パーキンソン病のモデルとしてドーパミン作動性ニューロンの神経変性を評価するために線虫を使用する方法を示します。さらに、遺伝子スクリーニングは、どちら変性を高めたり、神経保護の要因を識別するために使用されます。

ビデオ: Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Ole Isacson: Development of New Therapies for Parkinson's Disease

Ole Isacson: Development of New Therapies for Parkinson&#039;s Disease

Ole Isacson gives a concise overview of Parkinsons's disease, its causes, therapeutic strategies, and advances in Parkinson's research.

オレIsacson：パーキンソン病に対する新しい治療法の開発

生物学

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

マイクロインジェクションを使用して安定したトランスジェニック線虫の世代

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Murine Model for Parkinson&#039;s Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

パーキンソン病モデルマウス：6 - OHドーパミンの病変から行動試験に

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

トランスジェニックの世代 C.エレガンスバイオリスティック変換による

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

の生産 C.エレガンス</emとRecombineering経由>トランスジーン galK選択可能なマーカー

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Chromosomics：同時に新規OctoChrome FISHアッセイを使用した全24ヒト染色体における数値と構造変化の検出

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

線虫における身体サイズ調節に関与する遺伝子をスクリーニングするための戦略を給餌RNA媒介干渉を用いた<em&gt; Cエレガンス</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

C. elegansの走化性アッセイ

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

培養胚のための方法<em&gt; C.虫</em&gt;細胞

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

上の細菌の影響を測定する<em&gt; C.エレガンス</em卵保持アッセイを用いて&gt;行動

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...