Madison Grant, Steven Foltz, Halie Zastre and Junna Luan provided technical assistance. Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under award number AR065077. The content is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health.

<ol>
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The author declares that she has no competing financial interests.

余分な樹脂は、プールされた凍結切片のコレクションが遅くなりますし、RNA単離に樹脂汚染を埋め込む増加させることができるので、この方法で最良の結果を達成するために、組織の凍結保存中に筋肉の下3分の1または半分に制限された樹脂を埋め込む保ちます。また、針の均質化中細心の注意は、収量を最大化し、針を目詰まりの可能性を最小限にすることが重要です。プロトコルは、針?...

この技術の目的は、単一の小さな骨格筋ソース組織からアクセスでき、そのような組織学および遺伝子発現など異なるモダリティ、ことで、複数の実験的な分析を行うことです。顕微鏡アプリケーションを注意深く凍結保存中に氷晶アーチファクトの形成を制限するように制御されなければならないサンプルの保存方法に最も敏感です。したがって、この方法の開発は、部分的に免疫蛍光顕微鏡法および遺伝子発現の両方のための原料物質分析として-140℃の液体窒素冷却2-メチルブタン浴中で樹脂を埋め込んで被覆された凍結前脛骨（TA）筋に基づいています。 多様な技術的アプローチのために同一の原料物質を使用する必要性は、左右の筋肉が異なる条件、実験の1つずつの制御を表す筋肉内注射に基づく実験のために特に重要です。例えば、筋肉再生の研究で一μSCLEは反対側の筋肉がビヒクル注射対照1として機能する広範な組織の損傷を引き起こす毒素を注射します。同様に、筋疾患の遺伝子治療研究は、典型的には、反対側2の空ベクター、無関係ベクターまたはビヒクル対照と比較する筋肉内注射による遺伝子治療ベクターの検証で始まります。したがって、別のアプリケーションに各TA筋を供給することは不可能です。 この問題に対処するための一般的な戦略は次のとおりです。私は）ii）の追加のマウスを使用すること、またはiii）は、各アプリケーションのための筋肉の一部を遮断するために、アプリケーションごとに異なる筋肉群を使用します。しかし、筋群の間に実質的な差異は、それが困難な別個のアプリケーションからのデータを比較することを可能にする、追加の動物は費用を増加させ、他の選択肢が存在する場合、不十分正当化されます。異なるアプリケーションを調達するために解剖した後に筋肉を割ると、最良の選択肢の私ですnは多くの場合。しかし、筋肉片は、しばしば均質2-5のために液体窒素または機械的な粉砕技術の下で粉砕を使用するには小さすぎます。筋肉は、細胞外マトリックス及び収縮タンパク質を充填した高度に構造組織であるため、不十分な機械的な均質化は、その後のDNA、RNAまたはタンパク質の低い収率をもたらします。ここでの詳細な方法は、複数のアプリケーションで使用するための1つのソース筋からの組織の少量を可能にし、凍結切片と針粉砕を含めることは、より良好なRNAの収量のための機械的均質化を向上させます。

<table border="0" cellpadding="0" cellspacing="0" width="637">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Cork</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">23420-708</td>
			<td style="width:259px;">Cut into small squares with a sharp blade.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Plastic coverslip</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:115px;">12-547</td>
			<td style="width:259px;">Used to orient the muscle during freezing.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Low temperature thermometer</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">89370-158</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">2-methylbutane</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:115px;">M32631-4L</td>
			<td style="width:259px;">Caution: hazardous chemical. Store in flammable cabinet.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Embedding resin: &#34;cryomatrix&#34;</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">6769006</td>
			<td style="width:259px;">Other embedding resins can be substituted for cryomatrix.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">Cryostat</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">microm HM550 with disposable blade carrier</td>
			<td style="width:259px;">Any working cryostat should be sufficient for the protocol.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Disposable cryostat blade</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">3052835</td>
			<td style="width:259px;">Use an appropriate blade or knife for the cryostat to be used.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:135px;">RNAse decontamination solution: &#34;RNase Zap&#34;</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">AM9780</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">Analytical balance</td>
			<td style="width:128px;">Mettler Toledo</td>
			<td style="width:115px;">XS64</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">Paint brush</td>
			<td style="width:128px;">Daler Rowney</td>
			<td style="width:115px;">214900920</td>
			<td style="width:259px;">Use to handle cryosections. Can be found with in stores with simple art supplies.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">Razor blade</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">55411-050</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">Microscope slide</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">48311600</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:135px;">RNA organic extraction reagent: TRIzol</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">15596026</td>
			<td style="width:259px;">Caution: TRIzol is a hazardous chemical. Note: Only organic extraction reagents are recommended for RNA extraction from skeletal muscle.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">18 gauge needle</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">BD305185</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">22 gauge needle</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">BD305155</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">26 gauge needle</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">BD305115</td>
			<td style="width:259px;">Optional. Can be used for a third round of sample trituration in the RNA extraction protocol.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">1 mL syringe</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">BD309659</td>
			<td style="width:259px;">For very high value samples, a luer-lok syringe is recommended (e.g. VWR BD309628).</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">1-bromo-3-chloropentane (BCP)</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:115px;">B9673</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">For 70% ethanol in DEPC water: 200 proof alcohol</td>
			<td style="width:128px;">Decon Laboratories, Inc.</td>
			<td style="width:115px;">+M18027161M</td>
			<td style="width:259px;">Mix 35 ml 200 proof alcohol + 15 mL DEPC water.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">For 70% ethanol in DEPC water: DEPC-treated water</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">AM9922</td>
			<td style="width:259px;">Mix 35 ml 200 proof alcohol + 15 mL DEPC water.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">RNA purification kit: PureLink RNA minikit</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">12183018A</td>
			<td style="width:259px;">Final steps of RNA preparation.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">DNase/Rnase-free water&#160;</td>
			<td style="width:128px;">Gibco</td>
			<td style="width:115px;">10977</td>
			<td style="width:259px;">DEPC-treated water can also be used.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Spectrophotometer: Nanodrop 2000</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">NanoDrop 2000</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">Dnase I</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">AM2222</td>
			<td style="width:259px;">Treat purified RNA to remove any DNA contamination before downstream appications.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Hydrophobic pen</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">8899</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:135px;">Dulbecco&#39;s PBS</td>
			<td style="width:128px;">Gibco</td>
			<td style="width:115px;">14190</td>
			<td style="width:259px;">PBS for immunofluorescence protocol.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:135px;">Donkey serum</td>
			<td style="width:128px;">Jackson ImmunoResearch Laoratories, Inc</td>
			<td style="width:115px;">017-000-121</td>
			<td style="width:259px;">Rehydrate normal donkey serum stock according to the manufacturer&#39;s instructions, then dilute an aliquot to 5% for immunofluorescence.&#160; Normal goat serum can also be used.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:135px;">eMHC antibody</td>
			<td style="width:128px;">University of Iowa Developmental Studies Hybridoma Bank</td>
			<td style="width:115px;">F1.652</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Collagen VI antibody</td>
			<td style="width:128px;">Fitzgerald Industries</td>
			<td style="width:115px;">#70R-CR009x</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Donkey anti-rabbit AlexaFluor488</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">A21206</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Goat anti-mouse IgG1 AlexaFluor546</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td>A21123</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">DAPI (4&#39;,6-diamidino-2-phenylindole)</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">D1306</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">Aqueous mounting media: Permafluor</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">TA-030-FM</td>
			<td style="width:259px;">Only use mounting media designed for fluorescent applications with anti-fade properties.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Glass coverslip</td>
			<td style="width:128px;">VWR Scientific</td>
			<td style="width:115px;">16004-314</td>
			<td style="width:259px;">Use for mounting slides at the end of immunofluorescence protocl</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:135px;">Image analysis software: ImageProExpress</td>
			<td style="width:128px;">Media Cybernetics, Inc.</td>
			<td style="width:115px;">Image-Pro Express, or more advanced products</td>
			<td style="width:259px;">Freeware ImageJ should also work for manual counting. More advanced software with segmentation abiities may allow partial automation of the process.&#160; E.g. ImageProPremier.&#160;&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:135px;">Merge and map section images: Photoshop</td>
			<td style="width:128px;">Adobe</td>
			<td style="width:115px;">Photoshop</td>
			<td style="width:259px;"></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:135px;">Cardiotoxin</td>
			<td style="width:128px;">Sigma</td>
			<td style="width:115px;">C9659</td>
			<td style="width:259px;">Sigma C9659 has been discontinued. Other options for cardiotoxin are EMD Millipore #217503; American Custom Chemicals Corp. # BIO0000618; or Ge Script # RP17303; but these have not been validated.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:135px;">reverse transcription kit: Superscript III First-strand synthesis system</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">18080051</td>
			<td style="width:259px;">Any validated, high quality reverse transcription reagents can be used.</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;">Standard PCR: GoTaq Flexi polymerase system</td>
			<td style="width:128px;">Promega</td>
			<td style="width:115px;">M8298</td>
			<td style="width:259px;">Any validated, high quality Taq polymerase system can be used. If DNA sequencing is to be used for any application downstream of the PCR, then a high fidelity PCR system should be used instead.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:135px;">SYBR green</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:115px;">S7585</td>
			<td style="width:259px;">For use in qPCR when not using a dedicated qPCR master mix. Use with SuperROX (for Applied Biosystems instruments) and GoTaq Flexi polymerase and buffers.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:135px;">ROX: SuperROX, 15 mM</td>
			<td style="width:128px;">BioResearch Technologies, Inc. Novato CA</td>
			<td style="width:115px;">SR-1000-10</td>
			<td style="width:259px;">SuperROX is more stable in the PCR reaction, so it is preferred for use as a qPCR passive reference dye over ROX (carboxy-X-rhodamine). For qPCR with Applied Biosystems instruments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:135px;">Real-time PCR</td>
			<td style="width:128px;">Applied Biosystems</td>
			<td style="width:115px;">7900HT</td>
			<td style="width:259px;"></td>
		</tr>
	</tbody>
</table>

cryosectioning of contiguous regions of a single mouse skeletal muscle for gene expression and histological analyses

この方法では、連続した凍結切片は、単一のマウス骨格筋からの隣接領域を用いて、遺伝子発現のための組織の組織学とRNAの濃縮のための両方の顕微鏡アプリケーションを可能にするために収集されます。典型的には、それがバッファボリュームは、効率的な研削用途には低すぎるかもしれないので、まだ十分な機械的破壊することなく、小さな骨格筋サンプルの適切な均質化を達成するために挑戦され、緩衝剤の筋限界浸透の密集組織構造は、最終的に低いRNA収量を引き起こします。ここで報告されたプロトコルに従うことにより、30μmのセクションでは、収集され、バッファへの浸透のために露出表面積を増加させ、凍結切片およびその後の針均質化が筋肉を混乱させる機械的にできるようにプールしました。技術の主要な制限は、クライオスタットを必要とすることであり、それは比較的低いスループットです。しかし、高品質のRNAをプールし、Mの小さなサンプルから得られますuscle凍結切片は、多くの異なる骨格筋および他の組織のために、この方法は、アクセス可能にします。測定は直接実験的不確実性を低減し、のための小さな組織を供給するのに必要な複製の動物実験を低減するためにアプリケーション間で比較することができるように、また、この技術は、単一の骨格筋の隣接領域からの一致分析（ 例えば、組織の組織病理学および遺伝子発現）を可能にします複数のアプリケーション。

筋肉凍結切片のRNAは、品質が高く、ほとんどのアプリケーションのための十分な収量を提供します分析は、16骨格筋のRNA調製物を、8匹の対照マウスからプールした前脛骨（TA）筋の19.4〜41ミリグラムを用いて、 表1に示します。どちらの左（L）と右（R）TAの筋肉がメソッドを使用して、筋肉の損傷を引き起こす?...

Watch this Scientific Journal Video about Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses at JoVE.com

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

連続凍結切片を、単一のマウス骨格筋からの隣接領域を用いた遺伝子発現の測定のための組織学的応用およびRNAの濃縮を可能にするために収集されます。直接アプリケーション間で比較され、プールされた凍結切片と測定30mgの - 高品質のRNAが20から得られます。

遺伝子発現と組織学的分析のためにシングルマウス骨格筋の連続領域の凍結切片化

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

cryosectioning-contiguous-regions-single-mouse-skeletal-muscle-for

Research

JoVE Journal

Biology

15.5K ビュー.  University of Georgia. 連続凍結切片を、単一のマウス骨格筋からの隣接領域を用いた遺伝子発現の測定のための組織学的応用およびRNAの濃縮を可能にするために収集されます。直接アプリケーション間で比較され、プールされた凍結切片と測定30mgの - 高品質のRNAが20から得られます。 

ビデオ: Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

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

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 ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

大人と胚骨格筋のMicroexplantの文化と骨格筋幹細胞の単離

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

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 ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

成人骨格筋からの個々の筋線維とその衛星細胞の単離および培養

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

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 ...

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease.

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

組織トリアージや骨格筋疾患のモデルのために凍結

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...

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.