我々は、ノックインと成長している感覚突起のタイムラプスムービーを提供するための遺伝子のために構造を生成する際に彼の助けのために博士はマイケルベイカーに感謝したいと思います。我々はまた、空気銃の実験と技術支援のためにバージニアVandelinderに感謝します。

粒子は、dsRNA分子、DNAプラスミドや染料などの異なる試薬で被覆することができます。粒子の種類と直径が試薬と組織における標的細胞の深さに応じて選択される：より大きな粒子がさらに浸透するが、組織に何らかの損傷を与える可能性があります。 1。 dsRNAを被覆粒子の​​調製<ol><li>氷の上で100％イソプロパノールを置きます。 </li><li> 100ミリリットル結合バッファーで、再懸5 mgの金粒子（平均粒径は1.6μmS1600ri、シーシェル技術、LLC）。解決策の準備ができたら、次の手順に進みます。 </li><li>結合バッファーを100μlを添加して粒子溶液を希釈し、ボルテックスで軽く混和し、凝集を避けるために1-2分間超音波処理。 </li><li> dsRNAを/ siRNAの5 -10μgの（dsRNAは/ siRNAは〜1μg/μlの濃度に水に溶解する必要がある）を追加します。 </li><li> 2分間、室温でボルテックスし、休暇。 siRNAの飽和金粒子のこれらの濃度と時間間隔の結果。 </li><li>ペレット〜15秒のためのベンチトップ遠心機で〜2,500 rpmで遠心分離によるsiRNA導入/金微粒子複合体。 </li><li>上清を除去し、穏やかにペレットの最小限の中断で750 mlの冷イソプロパノールを加える。簡単にスピンし、上清を取り除く。 </li><li> 100μlの冷100％イソプロパノールで金粒子を再懸濁し、次に粒子の凝集体を分割する（2-3パルス）バスの超音波処理で簡単に超音波処理。最後に、スライドガラスの上に懸濁粒子を注ぎ、室温で乾燥させます。 </li></ol> 2。特定のアッセイのための実験セットアップの準備<ol><li>それぞれの銃は最高〜3mmまで〜50μmの最小値に至るまで、さまざまなノズルの開口径を持っています。銃のために使用される特定したがって、対象となる組織の面積に応じて選択されます。 </li><li>彼の圧力は、一般的にアップ〜100μmの（より高い圧力はより深い粒子を提供することができます）に、標的細胞の組織の深さに調整されます。胚とノズルの先端間の距離はまた、空気中の金粒子緩い勢いとして、浸透の深さに影響を与えます。 </li></ol> 3。プレコート粒子でガンをロードする粒子は、マニホールドに接続されているタイゴンチューブの粒子注入ラインに乾燥粉末としてロードする必要があります。 4℃で使用していない時は、乾燥雰囲気で保管してくださいこのチューブは、、同じ試薬と追加の実験で再利用することができます。私たちの実験のセットアップは、マニホールド内の別のポートを介してアッセイ当たり二つの異なる試薬を供給することができます。 <ol><li>タイゴンチューブのラインにそれらをロードする前にガラスのカバースリップまたはカミソリの刃のエッジを使用してスライドガラスから乾燥被覆粒子をこすり落とす。このタイゴンチューブは、クロスコンタミネーションを避けるためにのみこの試薬に固有になります。 </li><li>パーティクルをロードするように、全体チューブと集中に近いコネクタに沿って広がってからの粒子を保つために、コネクタの近くにU字状にチューブを曲げて。小さなヘラまたは折り畳んだ重量紙、及び胚の各シリーズのチューブに、それらの約半分の負荷を持つ粒子を拾う。均等に粒子を分散させるロードステップ中にチューブをタップします。 </li></ol> 4。動物の準備金粒子の配信の前後に、胚を室温で無菌人工池の水に保持されます。 <ol><li>シリコーンゴム（Sylgard 184、ダウコーニング、ミッドランド、MI）のフラット、5mmの厚切りに刻まれた溝に、上胚、腹側を置き、（無菌人工池の水の8％エタノール溶液中でそれらをanaesthetizeの段階で若い胚E6 - E8）は、実験を通してのないエタノールで人工池の水に残すことができます。 </li><li>すぐに粒子を撮影する前に、ソリューションの一部を除去することによって胚の腹側表面を明らかにする浴室のレベルを下げます。それを安定化させ、乾燥からその表面を保つために、その上に中央にカット小さな穴を持つ組織の一枚の紙を置きます。 </li></ol> 5。標的組織への被覆粒子の​​配信粒子の一つの負荷は通常、シングルショットでは通常、数百の粒子のために提供するとともに、最大10枚まで使用することができます。 <ol><li>銃に対応するチューブラインからの粒子のボーラス注入を引き起こして、0.3秒のための弁の1つを開くことにより、ショットを生成する。 </li><li>チューブ壁からの粒子を取り除くため、ガンの彼ストリームに彼らの注入を容易にするために、ショットの間静かにチューブをタップします。 </li><li>興味の組織の領域をカバーするために、複数のショットが必要になることがあります。 </li><li>粒子の配信後、マルチウェルプレートに1ウェルあたり好ましくは一つの胚、バック人工池の水の中に胚を置く。 </li></ol> 6。免疫染色および神経ラベリング RNAiのが（または異所性発現）が達成されるまで、粒子の配信後1​​〜3日間室温でと暗闇の中で胚を保つ。我々は、実験試料とコントロールに関する次のいずれかの手順を実行します。 <ol><li>ネトリンのmRNAの有無についてのアッセイにin situハイブリダイゼーション 。ジゴキシゲニン標識リーチネトリンのリボプローブは、全体のリーチの胚にハイブリダイズさせる。 </li><li>ネトリン蛋白質と神経組織（例えば、抗アセチル化チューブリンなど）に固有の他のタンパク質のアッセイに免疫細胞化学 。手続きは、全マウント胚でも実行されます。 </li><li>治療と制御セグメントへの関心のニューロン（私たちのケースでmechanosensory圧力ニューロン）の完全な喬木にラベルを付けるために色素が、いっぱいになるための単一の神経細胞への脂溶性色素 （DIIとDIO）、 のMicroinjections。このアッセイは、ライブ胚（無傷または切開）で実行されます。 </li></ol>

<ol>
	<li>Baker, M. W., Macagno, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAi+of+the+receptor+tyrosine+phosphatase+HmLAR2+in+a+single+cell+of+an+intact+leech+embryo+leads+to+growth-cone+collapse.">RNAi of the receptor tyrosine phosphatase HmLAR2 in a single cell of an intact leech embryo leads to growth-cone collapse.</a> Current Biology. 10, 1071-1074 (2000).</li><li>Baker, M. W., Macagno, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Hirudo+medicinalis+DNA+Promoters+for+Targeted+Gene+Expression.">Characterization of Hirudo medicinalis DNA Promoters for Targeted Gene Expression.</a> Journal of Neuroscience Methods. 156, 145-153 (2006).</li><li>Gan, W. B., 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=Cellular+expression+of+a+leech+netrin+suggests+roles+in+the+formation+of+longitudinal+nerve+tracts+and+in+regional+innervation+of+peripheral+targets.">Cellular expression of a leech netrin suggests roles in the formation of longitudinal nerve tracts and in regional innervation of peripheral targets.</a> Journal of Neurobiology. 40, 103-115 (1999).</li><li>Juven-Gershon, T., Cheng, S., Kadonaga, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+design+of+a+super+core+promoter+that+enhances+gene+expression.">Rational design of a super core promoter that enhances gene expression.</a> Nature Methods. 3, 917-922 (2006).</li><li>Rinberg, D., Simonnet, C., Groisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pneumatic+capillary+gun+for+ballistic+delivery+of+microparticles.">Pneumatic capillary gun for ballistic delivery of microparticles.</a> Applied Physics Letters. 87, 014103 (2005).</li><li>Shefi, O., Simonnet, C., Baker, M. W., Glass, J. R., Macagno, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microtargeted+gene+silencing+and+ectopic+expression+in+live+embryos+using+biolistic+delivery+with+a+pneumatic+capillary+gun.">Microtargeted gene silencing and ectopic expression in live embryos using biolistic delivery with a pneumatic capillary gun.</a> Journal of Neuroscience. 26, 6119-6123 (2006).</li></ol>

このデモでは、ノックインにライブリーチ胚の小さなローカライズされた体積のセルに金粒子を提供し、遺伝子をノックダウンする空気圧キャピラリー銃を使用。対象地域は、一般的に約150μmおよび粒子の直径は、0から50μmの間で調整可能だったの平均浸透深さ約〜15μmを、発見されたていた。粒子は軸索ガイダンス因子ネトリンのdsRNA分子でコーティングされたとき、彼らは明らかにだけ粒子を含む細胞で...

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<td>gold particles</td>
<td>Other</td>
<td>Seashell Technology, LLC</td>
<td>S1600ri</td>
<td>&nbsp;</td>
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<td>binding buffer</td>
<td>Reagent</td>
<td>Seashell Technology, LLC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>silicone rubber </td>
<td>Reagent</td>
<td>Dow Corning</td>
<td>Sylgard 184</td>
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localized rnai and ectopic gene expression in the medicinal leech

このビデオでは、我々は生きている組織への試薬の正確な微粒子銃配信のための空気圧キャピラリー銃の使用を示しています。我々は内部コントロールとして未処理のセグメントを残して、ヒル胚の選択されたセグメントに摂動の遺伝子発現パターンにプロシージャを使用します。 空気圧キャピラリー銃は、試料を開くことなく、開発の初期段階での細胞の内部の層に到達するために使用することができます。生体組織への物質のローカライズを導入する方法として、銃による遺伝子銃送達は、いくつかの利点があります：それは、高速、非接触および非破壊です。さらに、シングルキャピラリー銃は、異なる物質の独立した配信のために使用することができます。配達地域は、〜50から150ミクロンの横方向の寸法を持っていると、0〜50μmの間で調節可能である平均浸透深さ、周囲に15〜上程度を拡張できます。この配信は単一の細胞外注射を介して、または遺伝学的アプローチによるマイクロインジェクションし、全身のノックダウンによるノックダウンの間に中間の選択した場所にあるセルの限られた数を対象とすることができるという利点があります。 背側ドメインではないノックダウンまたは自然にある中央のニューロンによって、腹側体壁に表現されている軸索ガイダンス分子ネトリン、の式でノッキングの場合は、しかし、我々は、体壁にdsRNA又はプラスミド- DNAの分子を提供し、中枢神経節。 、（i）の特定のアッセイのための実験装置（加速圧力を調整する）の準備、（ⅱ）塗装ガンに被覆された粒子をロードするdsRNA又はDNAの分子と粒子、（III）：この手順は、次の手順が含まれます最大1アッセイ、（IV）の2つの試薬に（粒子の配信、（v）を標的組織（体壁または神経節）に被覆粒子の​​配信のために動物を準備し、そして（vi）の胚を処理して免疫染色、免疫組織化学と神経ラベリング）は2〜3日配達後、通常、結果を可視化する。 粒子がネトリンdsRNAをでコーティングされたとき、彼らは明らかに粒子のみを（通常は、細胞あたり1-2粒）を含む細胞内で発生したネトリン発現のノックダウン見える原因。粒子は、彼らが核で停止した神経細胞におけるプラスミドのエンコーディングEGFP誘起蛍光でコーティング。

Watch this Scientific Journal Video about Localized RNAi and Ectopic Gene Expression in the Medicinal Leech at JoVE.com

Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

このビデオでは、我々は新たな小型遺伝子銃を持つ生きている組織への試薬の正確な微粒子銃配信のための手順を示しています。我々は、単一セグメントの腹側体壁と神経節にdsRNAの分子を提供することで、ヒル胚における軸索ガイダンス分子ネトリンの発現をノックダウンされています。

ローカライズされたRNAiと創薬リーチにおける外来遺伝子の発現が

In this video, we show the use of a pneumatic capillary gun for the accurate biolistic delivery of reagents into live tissue. We use the procedure to ...

localized-rnai-and-ectopic-gene-expression-in-the-medicinal-leech

Research

JoVE Journal

Biology

9.5K ビュー.  University of California San Diego - UCSD. このビデオでは、我々は新たな小型遺伝子銃を持つ生きている組織への試薬の正確な微粒子銃配信のための手順を示しています。我々は、単一セグメントの腹側体壁と神経節にdsRNAの分子を提供することで、ヒル胚における軸索ガイダンス分子ネトリンの発現をノックダウンされています。

ビデオ: Localized RNAi and Ectopic Gene Expression in the Medicinal Leech

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

エレクトロポレーションのためのマウス骨髄からの一次造血細胞培養の準備

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

生物学

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

脊髄電気生理学

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

高効率で初代培養細胞をトランスフェクトするためにジーンパルサーMXcellのエレクトロポレーションシステムの使用

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

ロボット工学と植物組織における遺伝子発現の研究​​のための動的画像解析

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

ナマルバ細胞におけるIL - 4依存性遺伝子発現のsiRNAのノックダウン：遺伝子発現研究を簡素化する自動化セルカウンターの使い方

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

のパラフィン包埋して凍結切片ショウジョウバエアダルトの筋肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

アクティブT.の試験管内再構成でcastaneumのテロメラーゼ

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

RNAiは、Necromenic虫にマイクロインジェクションによる遺伝子ノックダウンとトランスジェネシスを媒介 Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

低酸素症後単一細胞レベルでのグローバルのRNA合成の分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.