精神的な支援のための美しいジェル用とトビアス·クラウスのウラKobalzを引き続き支援するためJanettビルケンフェルトの分野で熱狂的なサンプル収集のためのジルケキッパー、サラ·キーファー、マイケル·ワイスコニーBartschのとジルケフォークト·Heuckeに感謝、、。

すべての手順は、動物保護（TierSchG）のためのドイツの法律を遵守して実施された。 試薬および消耗品の1。準備<ol><li>分子グレードの水中5％（w / w）のキレックス-100溶液を調製する。標準1.5ミリリットルの反応管に200μLのアリコートを準備します。キレックス樹脂はサスペンションからの高速沈殿するように、一定分量の調製中絶えず再均質化するサスペンションが必要である。それは1つのバッチを50 ml以上を準備し、マグネチックスターラーを用いて均質化した懸濁液を維持することをお勧めです。キレックス溶液を周囲条件で年間保存することができる。 </li><li>試験対象の鳥のくちばしの幅よりも狭い幅のワットマン紙の部分をカット。用紙の長さは、サンプルを取るための方法によって異なります。 <ol><li>鉗子を使用して、作品は鳥のくちばしに挿入される鉗子ずにサンプル採取を可能にするのに十分な長さでなければなりません。として概算は、0.5cmのくちばしの長さ1cmの長さの一枚の紙は、問題ないはずです。 </li><li>鉗子を使用していない場合は、作品は長くなければなりませんが、一枚の剛性は、まだ上皮細胞の採取を可能にするはずである。 </li></ol></li><li>分析しようとするそれぞれのサンプルについて、PCRプレミックスの1アリコートを準備します。混合液25の全体積0.18のdNTPを含有する、2mMのMgCl 2、70mMのトリス-HCl、17.25 mMの硫酸アンモニウム、0.1％のTween-20、0.32μMのP2プライマー（TCTGCATCGCTAAATCCTTT）におけるPCR、0.32μMP8用プライマー（CTCCCAAGGATGAGRAAYTG）16および2単位のTaqポリメラーゼ。自家製のTaqポリメラーゼ17を使用することができる。 DNA溶液の最大量の添加を可能にするために、6μlのプレミックスは、数週間のために調製し、-20℃で保存することができる。 </li></ol> 2。サンプルの採取 PCRプライマーなどの鳥類の性決定がヒトDNAにアニーリングすることはできませんのために手袋を身に着けていることは必須ではない。のために他のジェノタイピングの目的は、それが賢明かもしれません。 <ol><li>興味のある鳥を捕獲し、そっと「リンゲルグリップ「18と1手などでそれを保持。鳥を圧迫しないようにしてください。 </li><li>鉗子でワットマン紙を持って、頬の内側、舌と後鼻孔を渡ってそれを数回強打。通常、サンプルは、30秒未満以内に得られる。 <ol><li>成体動物：種によって、そのくちばしを開くために鳥を得ることは困難かもしれません。通常、くちばしの側面に触れ、穏やかな圧力を適用すると、動作します。 </li><li>雛：孵化やヒナからのサンプリングがその物乞い行動はくちばしの内部への容易なアクセスを提供するので、この技術を用いて、特に簡単です。そのnestboxが開かれたとき、彼らはすぐに例を請うようにそれも、すべてでそれらを処理せずにサンプルを得ることができるかもしれません。 </li></ol></li><li>直ちに調製した反応管contaiに用紙を格納5％（w / w）のキレックス-100溶液200μlを寧。 </li></ol>あなたも/予定の場合はセクション6で説明したようにここで行い、雛をマークする必要があります。 DNA抽出前のサンプルの3。ストレージ<ol><li> -20℃での研究室で働いている場合は、サンプルの保存これは、周囲温度で保存サンプル（フィールド内など ）が可能かは難しいことではありません場合。 </li><li>試料を室温から-20℃の範囲の温度で3年以上保存することができる</li></ol> 4。DNA抽出<ol><li>試料が凍結している場合、室温で解凍。 </li><li>一枚の紙がキレックス-100溶液に沈め管の底にあることを確認します。 </li><li> 56℃で15分間、サンプルをインキュベート</li><li>簡単にボルテックスチューブの内容をスピンダウン。 </li><li> 100℃で8分間のサンプルをインキュベート</li><li> 3分間15,000×gでサンプルをスピン。 </li><li> Sを使用してくださいその後の遺伝子型判定のためupernatant。 </li></ol> 5。分子性決定<ol><li>サンプルあたり6μlのプレミックスプラス2の負のための追加のチューブ（必須）および陽性コントロール（オプション）を1 PCRチューブを準備します。ルーチンの性決定のポジティブコントロールとして、最後に成功した雌雄鑑別からのサンプルが含まれています。 </li><li> DNA抽出からのPCRプレミックスに上清の19μLを加える。キレックスビーズのキャリーオーバーを避けるために、溶液の表面からピペットにしてください。キレックスは陽イオン交換体であるため、深刻なすべての酵素反応を阻害するので、これは非常に重要です。 </li><li> 3分間、72℃で45秒の伸長ステップ、続いて55℃で30秒のアニーリング工程、続いて30秒間溶融工程94°C、45サイクルの各94℃でインキュベーション：以下のPCRプログラムを実行するC.最終チェースオフ工程では、反応は72℃で5分間インキュベートする</li><li> 2％標準TBEまたはTAE agaroを準備<html> PCR産物を分離するためのそれ自体のゲル。 </li><li>適切な電圧設定でアガロースゲルを実行します。 </li><li> UV光下でバンドを可視化し、写真を撮る。メスから発信サンプル中の2バンドが十分に分離されていることを確認します。 </li><li> 1（男性）または2（メス）のバンドの存在によって、女性のサンプルの男性を区別する。 </li></ol> 6。雛マーキング<ol><li>種/研究のための適切なスケジュールで、新たに孵化した雛のための巣を確認します（ほとんどのヒナが孵化した後のように朝には例えば 、毎日のチェックがお勧めします）。 </li><li>異なる特性の場所で巣内の各ひよこのダウン羽を切った。キンカチョウで浮き沈みの房は、雛の体全体で4つの特徴と異なる領域（ 図4A）で成長する。それぞれのひよこのための1の領域（ 図4B）をカット。つ以上の雛一ネスト内に存在する場合、固有の4 &#39;髪型&#39;は組み合わせることができる。 </li></ol>キンカチョウの場合ove_content &quot;&gt;：羽は検出が困難になり、雛サイズが鳴って許可した場合ダウン孵化ポスト10日にレッグバンドを適用します。

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J., 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=Buccal+swabs+as+a+reliable+source+of+DNA+for+sexing+young+and+adult+Common+Swifts+(Apus+apus).">Buccal swabs as a reliable source of DNA for sexing young and adult Common Swifts (Apus apus).</a> J. Ornithol. 153, 991-994 (2012).</li><li>Marion, W. R., Shamis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annotated-Bibliography+of+Bird+Marking+Techniques.">Annotated-Bibliography of Bird Marking Techniques.</a> Bird Banding. 48, 42-61 (1977).</li><li>Scharff, C., Adam, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenetics+of+birdsong.">Neurogenetics of birdsong.</a> Curr. Opin. Neurobiol. , (2012).</li><li>Griffith, S. C., Buchanan, K. 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著者らは、開示することは何もありません。

キレックスを用いた口腔スワブから雌雄鑑別は非常に高い成功率を示した。脚バンディングが可能になるまで切削雛のダウンの羽毛は、雛の間で差別化を可能にしました。 口腔スワブからのキレックス-DNA抽出に成功し、分子性判別を実行するための十分なDNAが得られた。性的二型羽によって検証としての性決定は正しい100％だった。ここで報告された成功率は2これまで...

個人認識、性判別およびジェノタイピングは、実験的研究の様々な基本的な前提条件である。 DNAの軸受材料を得て、（でも早い年齢で）明確に被写体をマーキング生理、行動、生存への影響を最小限に抑える必要があります。可能な限り、侵襲的処置はRRR原理1によれば避けるべきである。 非侵襲的な方法だけでな​​く、動物のために有益であるだけでなく、動物が少ない治療法の影響を受けているようにして得られたデータを向上することがあります。 鳥類では、DNAの性判別が糞2、羽毛3,4または口腔スワブ3,5,9などの非侵襲的に得られる材料の数に基づいて行うことができる。彼らは実行が容易であるために関係なく、患者の状態や年齢頬スワブの鳥類の雌雄鑑別のための選択の方法はほとんど失敗しないと取り扱いが短い、、である。 口腔スワブからこれまでのところ、DNAいずれかの市販のキット3,6又は時間3,6-8標準的なDNA抽出プロトコールを消費で抽出した。キットだけでなく、かなり高価ですが、彼らのプロトコルは、フィールドワークのための課題を課すことができる。いくつかの手続きの詳細、 例えば乾燥およびサンプルのインキュベーションは、フィールドには実用的ではない。特に実験プロトコルは、早ければ数分後孵化などからセックス依存の治療を必要とする設定で、結果を得るために、迅速な非侵襲的、信頼性が高く、簡単な方法のために促すとされている。 鳥類の分類群間でマーキング個人のためのかなりのツールボックスは、10を開発してきた。利用可能な技術の広い配列は、研究目的、種や予算のさまざまなを占めています。しかし、小さな雛をマークすると、新たな課題を研究者に直面しています。いくつかの種（ 例えばスズメ目）のヒヨコは、レッグバンドを適用するには小さすぎる、代替方法を必要とする親の子孫の動作を変更しないでください。フィールドと実験室での研究で動物福祉や技術の改善に意識や関心が高まっているように、非侵襲性の技術を使用することを強く奨励して好ましい。 このプロトコルは、個々に、レッグバンドを適用する前に、非常に若い雛をマークするために非侵襲的、迅速かつ容易に認識永続方法を提供することは可能である。このマーキング方法は、最も重要な鳥類の実験室モデルの種の一つ、ゼブラフィンチ（Taeniopygia滴状 ）11月13日に導入されている。プロトコルは、個々のマーキング技術10のための以前に発表され目標のすべてに準拠しており、すでに正常14,15を適用されている。

<table border="0" cellpadding="0" cellspacing="0" width="702">
	<tbody>
		<tr height="75">
			<td height="75" style="height:75px;width:191px;">Whatman 3MM Chromatography paper</td>
			<td style="width:163px;">e.g. Fisher Scientific</td>
			<td style="width:269px;">No.:3030-153</td>
			<td style="width:80px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:191px;">Chelex 100 Resin</td>
			<td style="width:163px;">Biorad</td>
			<td style="width:269px;">#143-2832</td>
			<td></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:191px;">Taq polymerase</td>
			<td style="width:163px;">addgene</td>
			<td style="width:269px;">http://www.addgene.org/25712/</td>
			<td></td>
		</tr>
		<tr height="75">
			<td height="75" style="height:75px;width:191px;">leg band (Flexi number rings for birds, diameter 2,5 mm)</td>
			<td style="width:163px;">Horst Stengel &#38; Sohn</td>
			<td style="width:269px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

who is who? non-invasive methods to individually sex and mark altricial chicks

多くの実験は、初期の子孫の性別を決定するだけでなく、初期の個体識別のための新生児のマーキングが必要です。該当するときはいつでも、動物福祉のガイドラインによれば、非侵襲的な技術が優先されるべき。私たちのグループでは、研究室で、現場での曲の鳥の異なる種で動作し、我々が正常なセックスに非侵襲的方法を適用し、個別に雛をマーク。本論文では、総合的な非侵襲的なツールボックスが表示されます。鳥を雌雄鑑別することは前に第二次性徴形質発現をPCRのためのDNA含有物質の収集を必要とします。私たちは、口腔スワブからDNAを抽出することにより、あらゆる年齢（ポスト孵化）の性鳥への迅速かつ簡単な方法を確立した。結果は、3時間以内に得ることができる。のための個々のマーキングひよこのダウン羽が孵化順序内での高速同定を可能にする特定のパターンでトリムされます。メソッドのセットは、標準装備の研究室でも容易に適用され、働いに特に適し特別な機器などの分野でのGはサンプリングと保存のために必要とされている。雛の取扱いを最小限にし、マーキングおよび性判別技術は、非侵襲的、それによって動物福祉ガイドラインのRRR原理を支援しています。

<html>口腔スワブは小鳥種々の性決定のためのDNAを得るために使用することができるサンプルは、キンカチョウ（Taeniopygia滴状、99の個人、年齢0日- 5歳）から採取した、カナリア諸島（Serinusのカナリア ）、ベンガルフィンチ（Lonchura線条体 ）、ナイチンゲール（ サヨナキドリ ）、シジュウカラ（ シジュウカラ ）とブラックバーズ（ ツ?...

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Who is Who? Non-invasive Methods to Individually Sex and Mark Altricial Chicks

このプロトコルは、孵化直後にマーキング極めて迅速、簡単、非侵襲的、信頼性の高い鳥の低コスト、分子性決定とそれらの非侵襲的、迅速、安全かつ容易に認識可能にする方法の便利なセットを提供します。雛の限られた処理が必要となります。メソッドのこの便利なツールボックスは、RRR-ガイドラインに完全に準拠しています。

誰が誰ですか？非侵襲的な方法に個別にセックス·アンド·ザ·マーク·晩成性の雛

Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal ...

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Biology

18.2K ビュー.  Freie Universität Berlin. このプロトコルは、孵化直後にマーキング極めて迅速、簡単、非侵襲的、信頼性の高い鳥の低コスト、分子性決定とそれらの非侵襲的、迅速、安全かつ容易に認識可能にする方法の便利なセットを提供します。雛の限られた処理が必要となります。メソッドのこの便利なツールボックスは、RRR-ガイドラインに完全に準拠しています。 

ビデオ: Who is Who? Non-invasive Methods to Individually Sex and Mark Altricial Chicks

Labeling hESCs and hMSCs with Iron Oxide Nanoparticles for Non-Invasive in vivo Tracking with MR Imaging

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative medicine. A non-invasive method to monitor the transplanted stem cells repeatedly in vivo would greatly enhance our ability to understand the mechanisms that control stem cell death and identify trophic factors and signaling pathways that improve stem cell engraftment. MR imaging has been proven to be an effective tool for the in vivo depiction of stem cells with near microscopic anatomical resolution. In order to detect stem cells with MR, the cells have to be labeled with cell specific MR contrast agents. For this purpose, iron oxide nanoparticles, such as superparamagnetic iron oxide particles (SPIO), are applied, because of their high sensitivity for cell detection and their excellent biocompatibility. SPIO particles are composed of an iron oxide core and a dextran, carboxydextran or starch coat, and function by creating local field inhomogeneities, that cause a decreased signal on T2-weighted MR images. This presentation will demonstrate techniques for labeling of stem cells with clinically applicable MR contrast agents for subsequent non-invasive in vivo tracking of the labeled cells with MR imaging.

For the evaluation of new stem cell therapies it is important to non-invasively track the injected cells in vivo. This video will show you how to label human mesenchymal and embryonic stem cells with iron oxide based contrast agents in vivo for subsequent MR imaging in vivo.

MRイメージングと生体内追跡非侵襲のための酸化鉄ナノ粒子で標識ヒトES細胞とhMSCs

In recent years, stem cell research has led to a better understanding of developmental biology, various diseases and its potential impact on regenerative ...

生物学

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

光イメージングによる非侵襲的検出のための蛍光色素で標識幹細胞

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Non-invasive 3D-Visualization with Sub-micron Resolution Using Synchrotron-X-ray-tomography

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard cuticle which makes it difficult to use protocols of classical histology. In addition, histological sectioning destroys the sample and can therefore not be used for unique material. Hence, a non-destructive method is desirable which allows to view inside small samples without the need of sectioning.
We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows us to reconstruct the internal organization in its natural state without the artefacts produced by histological sectioning. These date can be used for quantitative morphology, landmarks, or for the visualization of animated movies to understand the structure of hidden body parts and to follow complete organ systems or tissues through the samples.

We used synchrotron X-ray tomography at the European Synchrotron Radiation Facility (ESRF) to non-invasively produce 3D tomographic datasets with a pixel-resolution of 0.7&micro;m. Using volume rendering software, this allows the reconstruction of internal structures in their natural state without the artefacts produced by histological sectioning.

シンクロトロンX線トモグラフィを用いたサブミクロン分解能を持つ非侵襲的3次元可視化

Little is known about the internal organization of many micro-arthropods with body sizes below 1 mm. The reasons for that are the small size and the hard ...

A Magnetic Tether System to Investigate Visual and Olfactory Mediated Flight Control in Drosophila

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. As such, visual stabilization of upwind tracking directly aids in odor tracking. But do olfactory signals directly influence visual tracking behavior independently from wind cues? Also, the recent deluge of research on the neurophysiology and neurobehavioral genetics of olfaction in Drosophila has motivated ever more technically sophisticated and quantitative behavioral assays. Here, we modified a magnetic tether system originally devised for vision experiments by equipping the arena with narrow laminar flow odor plumes. A fly is glued to a small steel pin and suspended in a magnetic field that enables it to yaw freely. Small diameter food odor plumes are directed downward over the fly s head, eliciting stable tracking by a hungry fly. Here we focus on the critical mechanics of tethering, aligning the magnets, devising the odor plume, and confirming stable odor tracking.

Here we describe how to tether a fly in an olfactory magnetic-tether (OMT) apparatus. We describe how to align the rare-earth magnets and odor ports, and how to set mass flow rates for both the stimulus delivery and vacuum suction to achieve optimal odor tracking.

ショウジョウバエの視覚と嗅覚を介したフライトコントロールを調査するために磁気テザーシステム

It has been clear for many years that insects use visual cues to stabilize their heading in a wind stream. Many animals track odors carried in the wind. ...

Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells

This video demonstrates Shidham's method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually scattered cells and small cell groups. This technique uses HistoGel (Thermo Scientific) with conventional laboratory equipment.
The use of cell block sections is a valuable ancillary tool for evaluation of non-gynecologic cytology. They enable the cytopathologist to study additional morphologic specimen detail including the architecture of the lesion. Most importantly, they allow for the evaluation of ancillary studies such as immunocytochemistry, in-situ hybridization tests (FISH/CISH) and in-situ polymerase chain reaction (PCR). Traditional cell block preparation techniques have mostly been applied to non-gynecologic cytology specimens, typically for body fluid effusions and fine needle aspiration biopsies.
Liquid based cervicovaginal specimens are relatively less cellular than their non-gynecologic counterparts with many individual scattered cells. Because of this, adequate cellularity within the cell block sections is difficult to achieve. In addition, the histotechnologist sectioning the block cannot visualize the level at which the cells are at the highest concentration. Therefore, it is difficult to monitor the appropriate level at which sections can be selected to be transferred to the glass slides for testing. As a result, the area of the cell block with the cells of interest may be missed, either by cutting past or not cutting deep enough. Current protocol for Shidham's method addresses these issues. Although this protocol is standardized and reported for gynecologic liquid based cytology specimens, it can also be applied to non-gynecologic specimens such as effusion fluids, FNA, brushings, cyst contents etc for improved quality of diagnostic material in cell block sections.

Shidham's method for preparation of cell blocks with AV-marker from cytology specimens containing individually scattered cells and small cell groups.

個別散在セルの優位を持つ細胞診標本からのセルブロックの準備

This video demonstrates Shidham's method for preparation of cell blocks from liquid based cervicovaginal cytology specimens containing individually ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

遺伝子誘導の運命のマッピングへの実践的アプローチ：マークとトラックの細胞へのビジュアルガイドインビボでの</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

A Reversible, Non-invasive Method for Airway Resistance Measurements and Bronchoalveolar Lavage Fluid Sampling in Mice

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated procedures to obtain such measurements in the same animal are generally not feasible.  Here, we demonstrate protocols for obtaining from mice repeated measurements of AHR and bronchoalveolar lavage fluid samples.  Mice were challenged intranasally seven times over 14 days with a potent allergen or sham treated.  Prior to the initial challenge, and within 24 hours following each intranasal challenge, the same animals were anesthetized, orally intubated and mechanically ventilated.  AHR, assessed by comparing dose response curves of respiratory system resistance (RRS) induced by increasing intravenous doses of acetylcholine (Ach) chloride between sham and allergen-challenged animals, were determined.  Afterwards, and via the same intubation, the left lung was lavaged so that differential enumeration of airway cells could be performed.  These studies reveal that repeated measurements of AHR and BAL fluid collection are possible from the same animals and that maximal airway hyperresponsiveness and airway eosinophilia are achieved within 7-10 days of initiating allergen challenge.  This novel technique significantly reduces the number of mice required for longitudinal experimentation and is applicable to diverse rodent species, disease models and airway physiology instruments.  

Repeated measurements of rodent respiratory physiology and sampling of airway inflammatory cells are desirable, but generally not feasible. Here we describe a repeatable method for orally intubating mice that permits repeated measurements of airway hyperreactivity and sampling of airway inflammatory cells.

気道抵抗測定とマウスにおける気管支肺胞洗浄液のサンプリングのためのリバーシブル、非侵襲的方法

Airway hyperreactivity (AHR) measurements and bronchoalveolar lavage (BAL) fluid sampling are essential to experimental asthma models, but repeated ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

糞便グルココルチコイド分析：ウマにおける非侵襲性副腎モニタリング

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually untranslated &#8212; first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

代替プロモーターの使用およびマウスBcrp1の転写調節を発見する方法

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

Methods to Classify Cytoplasmic Foci as Mammalian Stress Granules

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to stress conditions, are implicated in various aspects of cell metabolism and survival. SGs modulate cellular signaling pathways, post-transcriptional gene expression, and stress response programs. The formation of these mRNA-containing granules is directly connected to cellular translation. SG assembly is triggered by inhibited translation initiation, and SG disassembly is promoted by translation activation or by inhibited translation elongation. This relationship is further highlighted by SG composition. Core SG components are stalled translation pre-initiation complexes, mRNA, and selected RNA-binding Proteins (RBPs). The purpose of SG assembly is to conserve cellular energy by sequestering translationally stalled housekeeping mRNAs, allowing for the enhanced translation of stress-responsive proteins. In addition to the core constituents, such as stalled translation preinitiation complexes, SGs contain a plethora of other proteins and signaling molecules. Defects in SG formation can impair cellular adaptation to stress and can thus promote cell death. SGs and similar RNA-containing granules have been linked to a number of human diseases, including neurodegenerative disorders and cancer, leading to the recent interest in classifying and defining RNA granule subtypes. This protocol describes assays to characterize and quantify mammalian SGs.

Stress Granules (SGs) are nonmembranous cytoplasmic structures that form in cells exposed to a variety of stresses. SGs contain mRNAs, RNA-binding proteins, small ribosomal subunits, translation-related factors, and various cell signaling proteins. This protocol describes a workflow that uses several experimental approaches to detect, characterize, and quantify bona fide SGs.

哺乳類のストレス顆粒としての細胞質叢を分類する方法

Cells are often challenged by sudden environmental changes. Stress Granules (SGs), cytoplasmic ribonucleoprotein complexes that form in cells exposed to ...