著者は、profを感謝。キアラカンパネッラとコンピテンスのアムラセンター、レーザーmicrodissectorの使用とそれらを提供するためのナポリフェデリコIIの大学、ナポリ、イタリア、、と3Dアニメーションの寛大な助けのためのMRヴィンチェンツォVicidomini。

パート1。特定とローカライズRNAiのを受けるショウジョウバエ成虫ウィングディスクの調製。 生体物質として、GAL4/UASシステム1によりローカライズと特定遺伝子サイレンシングにさらさトランスジェニック幼虫から得ショウジョウバエ成虫ウィングディスクを使わ。 GAL4ドライバ、第二UASレスポンダを運ぶOne：この幼虫子孫は2親系統が関与遺伝クロスからorginates。特定時間的及び/または空間パターンでGAL4を表現する加え、ドライバラインはGAL4発現ドメインを視覚化できるUAS - GFPレポーターを運びます。 UASレスポンダラインは、UASシーケンスの制御下、特に関心の選択遺伝子（呼び出しそれ遺伝子X）2をターゲットできなけれRNA（hpRNAを）サイレンシングヘアピンを表現。かつて細胞で発現、hpRNA Xは特に選択遺伝子を黙らできなけれ小さな干渉RNA（siRNAを）生成する切断さ。ドライバとレスポンダ導入遺伝子両方を運ぶ幼虫子孫、で、UAS -サイレンシングコンストラクトはのみGAL4タンパク質を発現それら細胞で転写、およびこうして選択遺伝子X.の空間的制限サイレンシングを誘導できなけれですさ可能さまざまなアプリケーション中でも、我々は縁をギザギザにした - GAL4の制御下たち関心の選択遺伝子（遺伝子X）（EN）ドライバ、翼ディスク3の後部コンパートメントで特定サイレンシングをトリガできます無音にこのアプローチを適用、テリトリーUAS - GFPトランスジーンの発現によってマークれました。 沈黙成虫翼ディスクは、手作業-解剖特に汚染組織、付着気管を取り巻く排除するケアを取っていた。各孤立翼ディスクはその後迅速かつ優しく選択区域の即時レーザーマイクロダイ用既治療、RNaseフリー解剖フレームへ移した。 パート2。翼ディスクの沈黙（後部）とunsilenced（前方）コンパートメントから選択した領域のレーザー顕微解剖。 とunsilenced（前方; GFP -非標識）コンパートメントが達成れた;いったん翼ディスクは沈黙（GFP標識後部）から特定領域のレーザー顕微解剖は、メンブレンにあった。このは容易両方に収まるエリア、ディスクのGFP陽性とGFP -負両側を描画によって行った。 microdissector下、まず選択周囲沿っカットを作る、GFP陽性組織上レーザービームを焦点。 microdissector選択スピードとパワーで切削進めます、しかし組織の選択面積が下チューブで下がるまでそれは、手動カッティングを絞り込むことです。このチューブはGFP陽性組織が後続RNA抽出の準備なるよう。、TRI試薬の小さな容積を含みます当社はその後unsilenced、GFP -負組織から同等領土を解剖ために、翼ディスクの反対、GFP -負側に切削領域を移さ。もう一度、自動カット後、とりあえず手動カットを精製に進んだ。カットが行われたいったん、またGFP -負組織はRNA抽出用準備、TRI試薬で回収した。 第3部。マイクロダイセクションティッシュエリアからRNA抽出と転写プロファイリング。 当社はキャップから液体をデタッチするチューブをスピンし1mlにTRI試薬を加算、その後標準TRI試薬プロトコル続くRNA抽出を行わ。十分材料を収集する、我々は100成虫翼ディスク上カッティング手順を繰り返した。約5000μm2である各の100分野から始まる、たち収量はRNA1.5μgのであった。マニュアル解剖の精度はその後ランダムヘキサ持つcDNAを合成スーパースクリプトIII（インビトロジェンを）用いた、RT - PCRによる沈黙とunsilencedサンプルでGFP発現をチェックによって制御れました。その規制経路の推定標的のそれらと共に、沈黙遺伝子Xの発現レベルを決定する焦点後続定量分析は、マスターミックス（インビトロジェンを）用いたRT - PCRリアルタイムによって行った。

<ol>
	<li>Elliott, D. A., Brand, A. H., Dahmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GAL4+System+A+Versatile+System+for+the+Expression+of+Gene.">The GAL4 System A Versatile System for the Expression of Gene.</a> , (2008).</li><li>Klein, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wing+disc+development+in+the+fly:+the+early+stages.+.">Wing disc development in the fly: the early stages. .</a> Current Opinion in Genetics &#38; Development. 11, 470-475 (2001).</li><li>Dietzl, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+transgenic+RNAi+library+for+conditional+gene+inactivation+in+Drosophila.">A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.</a> Nature. 448, 151-U1-151-U1 (2007).</li><li>Martin, F. A., Morata, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartments+and+the+control+of+growth+in+the+Drosophila+wing+imaginal+disc.">Compartments and the control of growth in the Drosophila wing imaginal disc.</a> Development. 133, 4421-4426 (2006).</li><li>Tabata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+morphogen+gradients.">Genetics of morphogen gradients.</a> Nature Reviews Genetics. 2, 620-630 (2001).</li><li>Moreno, E. &. a. m. p. ;. a. m. p., Basler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dMyc+transforms+cells+into+super-competitors.">dMyc transforms cells into super-competitors.</a> Cell. 117, 117-129 (2004).</li></ol>

unsilenced翼ディスクの前部/後部コンパートメントで達成されたそれらの基底レベルの発現量を基準にして、選択された遺伝子のXの活動は、サイレンシング時に40％に減少が見出された。対照的に、その推定ターゲット（遺伝子Y）のいずれかで、私たちはそれが否定的に遺伝子Xによって規制されているという仮説を検証するためにつながる、（7倍 - 増）が大幅にアップレギュレートを発見さ?...

株を飛ぶショウジョウバエUAS -サイレンシングラインはVDRCのRNAiコレクション2から取得することができます。 GAL4 -ドライバの行のコレクションはブルーミントンショウジョウバエストックセンター（インディアナ州、米国）から入手できます。 機器必要な機器は、レーザーmicrodissector（ライカLMD6000）、コールターマイクロ22R（ベックマン）、PCR（マイサイクラーBIO - RAD）、リアルタイムPCR（BIO - RAD iQ5）が含まれます。 ツール必要なツールが含まれます：ガラス井戸、ブラシ、マイクロダイセクション鉗子、ハンギングドロップスライド、注射針、PET膜、プラスチックチューブ（50ミリリットル、05ミリリットル、0.25 ml）を持つ金属製のフレームにマウントされている昆虫ピンを。

laser microdissection applied to gene expression profiling of subset of cells from the drosophila wing disc

組織の均質性は、ダウンストリームの解析を犠牲にすること、生体試料から生成することができる情報量の制限要因であることが証明されている。多くの組織内に存在する複雑で動的な細胞の関連を考えると、 生体の相互作用における徹底的な分子分析を再現するために、ひとつは、ネイティブのコンテキスト内で特定の細胞集団を分析することができなければなりません。レーザーを介したマイクロダイセクションでは、分子整合性を維持しながら、直接顕微鏡可視化の下で目的の細胞が明確に識別し、成功した収穫を可能にする、この目標を達成することができます。規模の大小、成長制御、細胞分化や器官形成を研究するために有利なモデルシステムを表す開発ショウジョウバエの翅のディスク、の定義の領域内の遺伝子発現を分析するためにこの技術を適用している。幼虫成虫ディスクは、早期に系統限定の境界によって前部と後部、背側と腹側の区画に分割されています。誘導GAL4 - UASバイナリ発現系を利用して、これらの区画の各々が特異的トランスジェニックで標識することができる構築GAL4 -ドライバ適切なの制御下にUAS - GFP遺伝子を発現するハエ。トランスジェニックディスクでは、細胞の離散部分集合の遺伝子発現プロファイリングは正確にレーザーカットを導くために蛍光GFPのシグナルを使用して、レーザーを介したマイクロダイセクションの後に決定することができます。 下流の様々なアプリケーションの中で、我々は、ローカライズされたRNA干渉（RNAi）の後にRNA転写物プロファイリングに焦点を当てた。 RNAi技術の出現により、GFP標識は、特定の遺伝子サイレンシングに離散的細胞集団の転写応答を決めるかもできるように、与えられた遺伝子のローカライズされたノックダウンと組み合わせて使用​​することができます。このアプローチを検証するために、我々は（GFPの発現で標識された）後からディスクに相当する領域を解剖し、それ以外の場合は遍在的に発現する遺伝子のP区画の地域サイレンシング時に前方（非標識）コンパートメント。 RNAは、マイクロダイセクション沈黙とunsilencedエリアと定量的リアルタイムRT - PCRによって決定される比較の遺伝子発現プロファイリングから抽出した。我々はこの方法が効果的にショウジョウバエ成虫のディスク内の細胞のサブセットの正確なトランスクリプトームに適用できることを示している。 RNAの源として巨大なディスクの準備が一般に細胞の均質性を前提としながら、実際、、それが転写発現は、位置情報の結果では、これらの構造の中で大きく変化することはよく知られています。レーザーカットを導くためにローカライズされた蛍光GFPのシグナルを使用して、より正確な転写解析を実施し、収益性の高い、ネイティブのコンテキスト内の異なる細胞系譜の転写産物プロファイリングなど、複数の異なるアプリケーションに適用することができる。

Watch this Scientific Journal Video about Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc at JoVE.com

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

レーザーマイクロダイセクションは、ローカライズされたRNAi実験に供ショウジョウバエの翅のディスクの特定のコンパートメントでの遺伝子発現プロファイリングの解析に適用した<em&gt; in vivoで</em&gt;。沈黙とunsilencedコンパートメントのと同等の領域から抽出したRNAは、ネイティブ組織のミクロ生態学のコンテキスト内での比較遺伝子発現プロファイルを決定する定量的RT - PCRにより解析した。

から細胞のサブセットの遺伝子発現プロファイリングへの応用レーザーマイクロダイセクションショウジョウバエウィングディスク

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

laser-microdissection-applied-to-gene-expression-profiling-subset

Research

JoVE Journal

Biology

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

11.1K ビュー.  University of Naples. レーザーマイクロダイセクションは、ローカライズされたRNAi実験に供ショウジョウバエの翅のディスクの特定のコンパートメントでの遺伝子発現プロファイリングの解析に適用した in vivoで。沈黙とunsilencedコンパートメントのと同等の領域から抽出したRNAは、ネイティブ組織のミクロ生態学のコンテキスト内での比較遺伝子発現プロファイル?...

ビデオ: Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

哺乳動物の組織のレーザーキャプチャーマイクロダイセクション

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

生物学

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 ...

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

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 ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

からのタグ付き核の親和性に基づく分離<em&gt;ショウジョウバエ</em遺伝子発現解析のための&gt;組織

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

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 ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

培養哺乳動物細胞内の生物学的経路のユビキチンおよびユビキチン様システムレギュレータを識別するために、siRNAのスクリーニング

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

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.