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。

<ol>
	<li>Russell, W. M. S., Burch, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+principles+of+humane+experimental+technique.">The principles of humane experimental technique.</a> , (1959).</li><li>Robertson, B. C., 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=Molecular+sexing+of+individual+kakapo,+Strigops+habroptilus+Aves,+from+feces.">Molecular sexing of individual kakapo, Strigops habroptilus Aves, from feces.</a> Mol. Ecol. 8, 1349-1350 (1999).</li><li>Yannic, G., 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=Description+of+microsatellite+markers+and+genotyping+performances+using+feathers+and+buccal+swabs+for+the+Ivory+gull+(Pagophila+eburnea).">Description of microsatellite markers and genotyping performances using feathers and buccal swabs for the Ivory gull (Pagophila eburnea).</a> Mol. Ecol. Resour. 11, 877-889 (2011).</li><li>Bello, N., 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=Isolation+of+genomic+DNA+from+feathers.">Isolation of genomic DNA from feathers.</a> J. Vet. Diagn. Invest. 13, 162-164 (2001).</li><li>Handel, C. M., 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=Use+of+buccal+swabs+for+sampling+DNA+from+nestling+and+adult+birds.">Use of buccal swabs for sampling DNA from nestling and adult birds.</a> Wildl. Soc. Bull. 34, 1094-1100 (2006).</li><li>Brubaker, J. L., 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=A+noninvasive,+direct+real-time+PCR+method+for+sex+determination+in+multiple+avian+species.">A noninvasive, direct real-time PCR method for sex determination in multiple avian species.</a> Mol. Ecol. Resour. 11, 415-417 (2011).</li><li>Arima, H., Ohnishi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Usefulness+of+avian+buccal+cells+for+molecular+sexing.">Usefulness of avian buccal cells for molecular sexing.</a> Ornithological Science. 5, 139-143 (2006).</li><li>Seki, S. -. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+sexing+of+individual+Ryukyu+Robins+Erithacus+komadori+using+buccal+cells+as+a+non-invasive+source+of+DNA.">Molecular sexing of individual Ryukyu Robins Erithacus komadori using buccal cells as a non-invasive source of DNA.</a> Ornithological Science. 2, 135-137 (2003).</li><li>Wellbrock, A. H. 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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zebra+Finch:+the+ultimate+Australian+supermodel.">The Zebra Finch: the ultimate Australian supermodel.</a> Emu. 110, 5-12 (2010).</li><li>Fee, M. S., Scharff, 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+songbird+as+a+model+for+the+generation+and+learning+of+complex+sequential+behaviors.">The songbird as a model for the generation and learning of complex sequential behaviors.</a> Ilar J. 51, 362-377 (2010).</li><li>Honarmand, M., 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=Stressful+dieting:+nutritional+conditions+but+not+compensatory+growth+elevate+corticosterone+levels+in+zebra+finch+nestlings+and+fledglings.">Stressful dieting: nutritional conditions but not compensatory growth elevate corticosterone levels in zebra finch nestlings and fledglings.</a> PLoS On. 5, (1371).</li><li>Krause, E. T., 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=Zebra+finch+nestlings+beg+more+under+better+nutritional+conditions.">Zebra finch nestlings beg more under better nutritional conditions.</a> Behaviour. 148, 1239-1255 (2011).</li><li>Griffiths, R., 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=A+DNA+test+to+sex+most+birds.">A DNA test to sex most birds.</a> Mol. Ecol. 7, 1071-1075 (1998).</li><li>Pluthero, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+purification+of+high-activity+Taq+DNA+polymerase.">Rapid purification of high-activity Taq DNA polymerase.</a> Nucleic Acids Res. 21, 4850-4851 (1993).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Joint+Working+Group+on+Refinement.+Laboratory+birds:+refinements+in+husbandry+and+procedures.+Fifth+report+of+BVAAWF/FRAME/RSPCA/UFAW+Joint+Working+Group+on+Refinement.">Joint Working Group on Refinement. Laboratory birds: refinements in husbandry and procedures. Fifth report of BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement.</a> Lab Anim. 35, 1-163 (2001).</li></ol>

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><tbody><tr><td>Whatman 3MM Chromatography paper</td><td>&lt;em&gt;e.g.&lt;/em&gt; Fisher Scientific</td><td>No.:3030-153</td><td></td></tr><tr><td>Chelex 100 Resin</td><td>Biorad</td><td>#143-2832</td><td></td></tr><tr><td>Taq polymerase</td><td>addgene</td><td>http://www.addgene.org/25712/</td><td></td></tr><tr><td>leg band (Flexi number rings for birds, diameter 2.5 mm)</td><td>Horst Stengel Sohn</td><td></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?...

Watch this Scientific Journal Video about Who is Who? Non-invasive Methods to Individually Sex and Mark Altricial Chicks at JoVE.com

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 ...

who-is-who-non-invasive-methods-to-individually-sex-mark-altricial

Research

JoVE Journal

Biology

18.3K 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

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, fluid and nutrient supply). Consequently both the nervous and vascular systems develop alongside each other and share striking similarities in their branching architecture. Here we report embryonic manipulations that allow us to study the simultaneous development of neural crest-derived nervous tissue (in this case the enteric nervous system), and the vascular system. This is achieved by generating chicken chimeras via transplantation of discrete segments of the neural tube, and associated neural crest, combined with vascular DiI injection in the same embryo. Our method uses transgenic chickGFP embryos for intraspecies grafting, making the transplant technique more powerful than the classical quail-chick interspecies grafting protocol used with great effect since the 1970s. ChickGFP-chick intraspecies grafting facilitates imaging of transplanted cells and their projections in intact tissues, and eliminates any potential bias in cell development linked to species differences. This method takes full advantage of the ease of access of the avian embryo (compared with other vertebrate embryos) to study the co-development of the enteric nervous system and the vascular system.

Dual Labeling of Neural Crest Cells and Blood Vessels Within Chicken Embryos Using ChickGFP Neural Tube Grafting and Carbocyanine Dye DiI Injection

Here we report dual labeling of neural crest cells and blood vessels using chickGFP neural tube intraspecies grafting combined with intra-vascular DiI injection. This experimental technique allows us to simultaneously visualize and study development of the NCC-derived (enteric) nervous system and the vascular system, during organogenesis.

鶏の胚内の神経堤細胞と血管の二重標識は、ひよこGFP 神経管移植とカルボシアニンDyI注射を用いた

All developing organs need to be connected to both the nervous system (for sensory and motor control) as well as the vascular system (for gas exchange, ...

発生生物学

Windowing Chicken Eggs for Developmental Studies

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling.  In addition, it allows for molecular perturbations of several types, including placement of protein-coated beads and introduction of plasmid DNA using in ovo electroporation.  These experiments have yielded important data on the development of the central and peripheral nervous systems.  For many of these studies, it is necessary to open the eggshell and reclose it without perturbing the embryo's growth.  The embryo can be examined at successive developmental stages by re-opening the eggshell.  While there are several excellent methods for opening chicken eggs, in this article we demonstrate one method that has been optimized for long survival times.  In this method, the egg rests on its side and a small window is cut in the shell.  After the experimental procedure, the shell is used to cover the egg for the duration of its development.  Clear plastic tape overlying the eggshell protects the embryo and helps retain hydration during the remainder of the incubation period.  This method has been used beginning at two days of incubation and has allowed survival through mature embryonic ages.

The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In this article we demonstrate one egg preparation method that has been optimized for long survival times.

発達研究のためのウィンドウ鶏卵

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has ...

生物学

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

クロス種の移植による神経堤の移行および分化の解析

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

神経科学

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

迅速かつ定量的なチック絨毛管内膜モデルを用いた転移性調節因子の発見

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

がん研究

In Ovo Feeding of Commercial Broiler Eggs: An Accurate and Reproducible Method to Affect Muscle Development and Growth

Within the past three decades, red meat and poultry scientists focused on developing strategies and technologies to manipulate muscle development during embryonic and fetal development. This area continues to be an area of focus because muscle fiber number is established during this time and determines the basis for all future growth. In poultry, numerous studies demonstrated in ovo feeding of growth factors, vitamins, or other nutrients improved chick embryonic muscle and intestinal development. Improving in ovo muscle development could benefit the poultry industry by possibly influencing meat yield, growth rate, or myopathy conditions. During the past five years, the Gonzalez Laboratory at the University of Georgia developed a nicotinamide riboside in ovo feeding methodology for broiler-chicken embryos, which altered&#160;muscle development. When injected into a developing embryo&#39;s yolk sac, nicotinamide riboside increased pectoralis major muscle weight and muscle fiber density at hatch. This protocol will demonstrate a methodology to accurately and reproducibly conduct in ovo feeding studies utilizing commercial standard- and high-yielding broiler embryos. These data and methods will allow other research groups to perform in ovo feeding studies with much success and reproducibility.

In Ovo Feeding of Commercial Broiler Eggs: An Accurate and Reproducible Method to Affect Muscle Development and Growth

A robust methodology has been developed for conducting in ovo feeding research trials utilizing unincubated commercial broiler eggs to test the ability of natural and synthetic compounds, in this case, nicotinamide riboside, to influence muscle development and growth.

オヴォで 市販のブロイラー卵の給餌:筋肉の発達と成長に影響を与える正確で再現可能な方法

Within the past three decades, red meat and poultry scientists focused on developing strategies and technologies to manipulate muscle development during ...

Analysis of Cell Differentiation, Morphogenesis, and Patterning During Chicken Embryogenesis Using the Soaked-Bead Assay

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of somatic cells, tissues, and organs. The precise activation of these genetic programs is regulated by morphogens, diffusible molecules that direct cell fate at different thresholds. Understanding how genetic activation coordinates morphogenesis requires the study of local interactions triggered by morphogens during development. The use of beads soaked in proteins or drugs implanted into distinct regions of the embryo enables studying the role of specific molecules in the establishment of digits and other developmental processes. This experimental technique provides information on the control of cell induction, cell fate, and pattern formation. Thus, this soaked bead assay is an extremely powerful and valuable experimental tool applicable to other embryonic models.

The soaked bead assay involves targeted delivery of test reagent at any developmental time point to study the regulation of cell differentiation and morphogenesis. A detailed protocol, applicable to any experimental animal model, for preparing three different types of soaked beads and implanting these in the interdigit of a chicken embryo is presented.

浸漬ビーズアッセイを用いたニワトリ胚発生時の細胞分化、形態形成、パターニングの解析

A multitude of genetic programs is activated during embryonic development that orchestrates cell differentiation to generate an astounding diversity of ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

ウズラ·アヒルキメラを使用して顔面発展に種特異的な貢献を評価する

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

Evaluation of Auditory Brainstem Response in Chicken Hatchlings

The auditory brainstem response (ABR) is an invaluable assay in clinical audiology, non-human animals, and human research. Despite the widespread use of ABRs in measuring auditory neural synchrony and estimating hearing sensitivity in other vertebrate model systems, methods for recording ABRs in the chicken have not been reported in nearly four decades. Chickens provide a robust animal research model because their auditory system is near functional maturation during late embryonic and early hatchling stages. We have demonstrated methods used to elicit one or two-channel ABR recordings using subdermal needle electrode arrays in chicken hatchlings. Regardless of electrode recording configuration (i.e., montage), ABR recordings included 3-4 positive-going peak waveforms within the first 6 ms of a suprathreshold click stimulus. Peak-to-trough waveform amplitudes ranged from 2-11 &#181;V at high-intensity levels, with positive peaks exhibiting expected latency-intensity functions (i.e., increase in latency as a function of decreased intensity). Standardized earphone position was critical for optimal recordings as loose skin can occlude the ear canal, and animal movement can dislodge the stimulus transducer. Peak amplitudes were smaller, and latencies were longer as animal body temperature lowered, supporting the need for maintaining physiological body temperature. For young hatchlings (&#60;3 h post-hatch day 1), thresholds were elevated by ~5 dB, peak latencies increased ~1-2 ms, and peak to trough amplitudes were decreased ~1 &#181;V compared to older hatchlings. This suggests a potential conductive-related issue (i.e., fluid in the middle ear cavity) and should be considered for young hatchlings. Overall, the ABR methods outlined here permit accurate and reproducible recording of in-vivo auditory function in chicken hatchlings that could be applied to different stages of development. Such findings are easily compared to human and mammalian models of hearing loss, aging, or other auditory-related manipulations.

We have used standard auditory brainstem response (ABR) techniques and applied them to hatchling chickens, a precocious avian model for auditory function. The protocol outlines animal preparation and ABR acquisition techniques in detail, with steps that could translate to other avian or rodent models.

ニワトリ孵化子の聴覚脳幹応答の評価

The auditory brainstem response (ABR) is an invaluable assay in clinical audiology, non-human animals, and human research. Despite the widespread use of ...

Visually Sexing Loggerhead Shrike (Lanius Ludovicianus) Using Plumage Coloration and Pattern

The loggerhead shrike is a small sexually monomorphic passerine bird using grassland habitats across North America. Based on Breeding Bird Survey data, the species has undergone a drastic decline since the mid-1960s. The cause of decline is unknown, and research is actively underway to address this knowledge gap. These efforts are hindered by an inability to sex the species in hand, which to date was only possible using molecular markers. Here, we present a protocol to sex loggerhead shrikes by visually analyzing the coloration and pattern in the sixth primary feather. The application of the method will facilitate our ability to identify threats on a finer scale than has been possible to date and to address various ecological and evolutionary hypotheses. The methodology is simple and results reliable&#8211;we encourage including this method for research of both in situ and ex situ populations.

Visually Sexing Loggerhead Shrike Lanius Ludovicianus Using Plumage Coloration and Pattern

We present a protocol to characterize the sex of loggerhead shrike visually based on the coloration and pattern of the sixth primary wing feather.

視覚的にセックスのロガーヘッド・シュライク (ラニウス・ルドヴィキアヌス) 羽毛の着色とパターンを使用

The loggerhead shrike is a small sexually monomorphic passerine bird using grassland habitats across North America. Based on Breeding Bird Survey data, ...

Basic Chick Care and Maintenance

Chicks (Gallus gallus domesticus) are a valuable research tool, not only for studying important concepts in vertebrate development, neuroscience, and tumor biology, but also as an efficient system in which to propagate viruses. Although eggs can be purchased from external suppliers and working with chicks requires very little specialized equipment, an understanding of proper handling procedures is required for normal embryo development.
This video will provide an overview of egg handling principles, including an explanation of the incubation parameters that can profoundly impact development: temperature, humidity, and egg rotation. Most experiments that use chicken eggs require access to the embryo within the shell, which is achieved by cutting a small, resealable hole, or &ldquo;window.&rdquo; This process is described in step-by-step detail, along with several other techniques essential for working with chicks, such as candling and India ink injection. Finally, the video will review some practical applications of these basic techniques in advanced scientific research.

Basic Care and Maintenance of Gallus gallus domesticus Chicks

ニワトリのケアとメンテナンスの基本

Chicks (Gallus gallus domesticus) are a valuable research tool, not only for studying important concepts in vertebrate development, neuroscience, and ...

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

要約

さらに動画を探す

この動画の章

鶏の胚内の神経堤細胞と血管の二重標識は、ひよこ^GFP神経管移植とカルボシアニンDyI注射を用いた

発達研究のためのウィンドウ鶏卵

クロス種の移植による神経堤の移行および分化の解析

迅速かつ定量的なチック絨毛管内膜モデルを用いた転移性調節因子の発見

オヴォで市販のブロイラー卵の給餌:筋肉の発達と成長に影響を与える正確で再現可能な方法

浸漬ビーズアッセイを用いたニワトリ胚発生時の細胞分化、形態形成、パターニングの解析

ウズラ·アヒルキメラを使用して顔面発展に種特異的な貢献を評価する

ニワトリ孵化子の聴覚脳幹応答の評価

視覚的にセックスのロガーヘッド・シュライク (ラニウス・ルドヴィキアヌス) 羽毛の着色とパターンを使用

ニワトリのケアとメンテナンスの基本

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

要約

さらに動画を探す

この動画の章

鶏の胚内の神経堤細胞と血管の二重標識は、ひよこGFP 神経管移植とカルボシアニンDyI注射を用いた

発達研究のためのウィンドウ鶏卵

クロス種の移植による神経堤の移行および分化の解析

迅速かつ定量的なチック絨毛管内膜モデルを用いた転移性調節因子の発見

オヴォで 市販のブロイラー卵の給餌:筋肉の発達と成長に影響を与える正確で再現可能な方法

浸漬ビーズアッセイを用いたニワトリ胚発生時の細胞分化、形態形成、パターニングの解析

ウズラ·アヒルキメラを使用して顔面発展に種特異的な貢献を評価する

ニワトリ孵化子の聴覚脳幹応答の評価

視覚的にセックスのロガーヘッド・シュライク (ラニウス・ルドヴィキアヌス) 羽毛の着色とパターンを使用

ニワトリのケアとメンテナンスの基本

鶏の胚内の神経堤細胞と血管の二重標識は、ひよこ^GFP神経管移植とカルボシアニンDyI注射を用いた

オヴォで市販のブロイラー卵の給餌:筋肉の発達と成長に影響を与える正確で再現可能な方法