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<html> 1。体の大きさと翼の大きさの変化を生成するために飼育し、食事の操作 合理的と概要。スケーリングの関係の表現、最終的な臓器や体の大きさの変化を作り出す発達規制プロセスで最終的にかかっています。これらのプロセスは、最善のようなD.のような（すなわち、完全変態）完全に変成昆虫を作品から説明されています器官は幼虫11,15-17に似たgrub内に未分化&quot;成虫ディスク&quot;として成長キイロ 、。 ショウジョウバエでは、最終的な臓器や身体の大きさは幼虫の段階で成長によって規制されている、彼らは、サード幼虫齢18の先頭に向かって羽化するための最小限の実行可能な重量（MVW E）に達するまで、幼虫は第1および第2の幼虫齢によって成長する。 MVW Eの達成前に飢餓が羽化を防止します。しかし、MVW Eで餓死している幼虫は、非常に小さなサイズではあるが、現実的な大人を生成します。これはため、開発中に栄養を減少さだけでなくハエではなく、ほとんどすべての動物19から21に、最終的な身体と器官の大きさを低減します。蛹化が発生する前にMVW Eが達成された後、食品の可用性を操作するが、ことによって、それが大幅に（例えば、個々の遺伝的変異）変異の他の情報源を上回るボディサイズに極端な表現型可塑性を誘発することが可能である。 <ol><li>卵は50のロットに分けて、表現型する母集団から採取し、標準的なフライ食品10mlのを含むバイアルに配置されます。産卵が継続的に発生するため、卵は3つの年齢コホートを生産、三日ごとに24時間を集めている。 </li><li>最古のコホートは、放浪の段階に達するか、単に蛹になるために開始されたときに、食事の操作が適用されます。この時点で、最古のコホートがよくMVWのEを超えている、最も若いコホートでは、非常にMVWのEに近いされ、残りのコホートでは、どこかにこれらの両極端の間にです。従って、この時点で食品の除去は、最古のコホートから、残りのコホートから最年少コホートとの中間サイズの大人からの非常に小さな大人を非常に大きな大人が生成されます。バイアルから幼虫を削除するには、約5ミリリットル40％ショ糖溶液のは、すべてのバイアルに追加されます。幼虫を解放するには、食べ物は中間の速度に設定された加振台上で15〜20分間撹拌する。 </li><li>浮遊幼生は、細かい（00）絵筆でショ糖溶液、湿った綿栓を含むバイアルに配置し、蛹になるために許可されてから削除されます。また、幼虫はこのようなメチルセルロース22のような非消化性食品添加物に配置することができます。 </li></ol> 2。体の大きさの推定 合理的と概要 。これまでの研究では、胸部の長さが全体のボディサイズ22の理想的なプロキシ未満であることを示している。その代わり、私たちは体の大きさの尺度として、蛹のサイズを使用してください。最大ボディサイズは、蛹化の前に摂食の停止に固定されています。その結果、蛹の大きさと成人の体のサイズ23との間の緊密な相関関係がある。 <ol><li>蛹は、顕微鏡用スライドガラスに腹側を上に配置し、解剖顕微鏡に接続されたデジタルカメラを用いて画像化している。各画像は、キャプチャ一意の識別コードを割り当てられ、コンピュータにデジタルカメラを接続し、ライブフィードを介してコンピュータに転送されます。 </li><li>画像形成された蛹は、その個々の固有のコードで標識したフライ食品の1mlを含む個々の2mlのepitubes、に移動し、ガス交換を可能にするためにairholesでパンクチャされています。 </li><li>蛹の大きさは蛹のシルエットのピクセル数として推定されている。他のサイズのメトリクスを用いることもできる。 </li></ol> 3。特性（翼）サイズの見積もり 合理的と概要 。イメージの翼に、我々は、&quot;ウインググラバー&#39;24、引き出しとその身体へのライブ、麻酔フライ垂直の翼を保持するを使用してください。ガラスの2枚の間に押された、これは翼が2つの次元のオブジェクトとしてイメージを作成することができます。 <ol><li>ハエはCO 2で軽く麻酔し、コンピュータに接続されているデジタルカメラを装着した解剖顕微鏡で翼グラバの位置に配置。 </li><li>翼は、その個々の固有のコードの下に画像化して保存されます。 </li><li>翼のサイズが限界固定ランドマークを外接または翼シルエットの画素数として最小凸多角形を使って推定することができます。 </li></ol> 4。スケーリングの関係の推定<ol><li>データは、コホート全体でプールが性別により分離し、対数変換されます。 </li><li>スケーリングの関係は、タイプII（すなわち、長軸の削減）回帰（12のレビューを参照）、各グループごとに推定されたパラメーターを使用してサイズのデータに適合している。 </li></ol> 5。代表的な結果： テント&quot;&gt;私たちの食生活の操作は、本体のサイズ（図1）と形質の大きさ（図2）の広い範囲を生成します。互いに対して対数変換特性をプロットすると、特定の生物学的なグループ内共分散の彼らのパターンを明らかにし、スケーリングの関係をフィッティング許可グループへ（図3）。ボディサイズと形状のスケールがグループ全体の大きさとどのように形状のcovariesの比較を可能にする方法について説明スケーリング関係のパラメータの抽出。 <img alt="図1" src="/files/ftp_upload/3162/3162fig1.jpg" /> 個人がMVWのEを達成した後、食事の操作を介して生成された粒度分布の極値から図1。蛹に。実際の蛹の画像は右の蛹の大きさの測定のためのそれらに対応するシルエットを左に表示されています。スケールバーは1mmです。 <img alt="図2" src="/files/ftp_upload/3162/3162fig2.jpg" /> 個人がMVWのEを達成した後、 図2。ウイングスは、ダイエットの操作を介して生成される体のサイズ分布の両極端から取得。実際の翼のイメージは、右記の翼大きさの測定のためのそれらに対応するシルエットを左側に表示されています。スケールバーは1mmです。 <img alt="図3" src="/files/ftp_upload/3162/3162fig3.jpg" /> 図3。性別によるキイロショウジョウバエのための翼胴サイズスケーリング。幼虫として1（灰色の円）または2日間（白丸）を餓死完全飼育（くま）、した各性別の個人のための対数変換したデータ。 II型の回帰直線は、それぞれの性別のために示されています。各年齢間のコホート、〜0.1ミリメートル（〜7％）の平均翼面積の減少と〜0.2とする。（〜8％）が観察された表現型の全範囲を増加させる、発生します。回帰は、男性が（傾き1.06）少しhyperallometricであるのに対し、女性はこのスケーリング関係（0.96の傾き）のための若干hypoallometricであることを明らかにする。

<ol>
	<li>Schmidt-Nielsen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Scaling: Why is animal size so important. , (1984).</li><li>Calder, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Size, function and life history. , (1984).</li><li>Huxley, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Problems of relative growth. , (1932).</li><li>Gould, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allometry+and+size+in+ontogeny+and+phylogeny.">Allometry and size in ontogeny and phylogeny.</a> Biol. Rev. 41, 587-640 (1966).</li><li>Alberch, P., Gould, S. J., Oster, G. F., Wake, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+and+shape+in+ontogeny+and+phylogeny.">Size and shape in ontogeny and phylogeny.</a> Paleobiology. 5, 296-317 (1979).</li><li>Thompson, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> On growth and form. , (1917).</li><li>Klingenberg, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterochrony+and+allometry:+The+analysis+of+evolutionary+change+in+ontogeny.">Heterochrony and allometry: The analysis of evolutionary change in ontogeny.</a> Biol. Rev. 73, 79-123 (1998).</li><li>Dubois, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sur+le+rapport+de+l'enc&#233;phale+avec+la+grandeur+du+corps+chez+les+Mammiferes.">Sur le rapport de l'enc&#233;phale avec la grandeur du corps chez les Mammiferes.</a> Bull. Soc. Anthropol. 8, 337-374 .</li><li>Huxley, J. S., Teissier, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Terminology+of+relative+growth.">Terminology of relative growth.</a> Nature. 137, 780-781 (1936).</li><li>Huxley, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constant+differential+growth-ratios+and+their+significance.">Constant differential growth-ratios and their significance.</a> Nature. 114, 895-896 (1924).</li><li>Shingleton, A. W., Frankino, W. A., Flatt, T., Nijhout, H. F., Emlen, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+and+shape:+The+developmental+regulation+of+static+allometry+in+insects.">Size and shape: The developmental regulation of static allometry in insects.</a> BioEssays. 29, 536-548 (2007).</li><li>Frankino, W. A., Emlen, D., Shingleton, A. W., Garland, T., Rose, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+approaches+to+studying+the+evolution+of+morphological+allometries:+The+shape+of+things+to+come.">Experimental approaches to studying the evolution of morphological allometries: The shape of things to come.</a> Experimental evolution: Concepts, methods, and applications. , (2009).</li><li>Emlen, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+selection+on+horn+length-body+size+allometry+in+the+horned+beetle+Onthophagus+acuminatus.">Artificial selection on horn length-body size allometry in the horned beetle Onthophagus acuminatus.</a> Evolution. 50, (1996).</li><li>Emlen, D. J., Nijhout, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+and+evolution+of+exaggerated+morphologies+in+insects.">The development and evolution of exaggerated morphologies in insects.</a> Annu. Rev. Entomol. 45, 661-708 (2000).</li><li>Shingleton, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body-size+regulation:+Combining+genetics+and+physiology.">Body-size regulation: Combining genetics and physiology.</a> Curr. Biol. 15, 825-827 (2005).</li><li>Nijhout, H. F., Davidowitz, G., Roff, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+analysis+of+the+mechanism+that+controls+body+size+in+Manduca+sexta.">A quantitative analysis of the mechanism that controls body size in Manduca sexta.</a> J. Biol. 5, 16.11-16.15 (2006).</li><li>Davidowitz, G., Nijhout, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physiological+basis+of+reaction+norms:+The+interaction+among+growth+rate,+the+duration+of+growth+and+body+size.">The physiological basis of reaction norms: The interaction among growth rate, the duration of growth and body size.</a> Integr. Comp. Biol. 44, 443-449 (2004).</li><li>Stieper, B. C., Kupershtok, M., Driscoll, M. V., Shingleton, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaginal+discs+regulate+developmental+timing+in+Drosophila+melanogaster.">Imaginal discs regulate developmental timing in Drosophila melanogaster.</a> Dev. Biol. 321, 18-26 (2008).</li><li>Colombani, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nutrient+sensor+mechanism+controls+Drosophila+growth.">A nutrient sensor mechanism controls Drosophila growth.</a> Cell. 114, 739-749 (2003).</li><li>Mirth, C. K., Riddiford, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+assessment+and+growth+control:+How+adult+size+is+determined+in+insects.">Size assessment and growth control: How adult size is determined in insects.</a> BioEssays. 29, 344-355 (2007).</li><li>Thissen, J. P., Ketelslegers, J. M., Underwood, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+regulation+of+the+insulin-like+growth+factors.">Nutritional regulation of the insulin-like growth factors.</a> Endocr. Rev. 15, 80-101 (1994).</li><li>Boots, M., Begon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resource+limitation+and+the+lethal+and+sublethal+effects+of+a+viral+pathogen+in+the+Indian+meal+moth,+Plodia+interpunctella.">Resource limitation and the lethal and sublethal effects of a viral pathogen in the Indian meal moth, Plodia interpunctella.</a> Ecol. Entomol. 19, 319-326 (1994).</li><li>Shingleton, A. W., Mirth, C. K., Bates, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+model+of+static+allometry+in+holometabolous+insects.">Developmental model of static allometry in holometabolous insects.</a> Proc. R. Soc. B. 275, 1875-1885 (2008).</li><li>Stern, D. L., Emlen, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developmental+basis+of+allometry+in+insects.">The developmental basis of allometry in insects.</a> Development. 126, 1091-1101 (1999).</li><li>Emlen, D. J., Allen, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotype+to+phenotype:+Physiological+control+of+trait+size+and+scaling+in+insects.">Genotype to phenotype: Physiological control of trait size and scaling in insects.</a> Integr. Comp. Biol. 43, 617-634 (2003).</li></ol>

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形態学的スケーリングは、ほぼ1世紀の間進化生物学の激しい研究の対象となっている。最近では、成長とスケーリングの規制の近接基礎（例えば、12,15,23-25 ​​のレビューを参照）の開発、生理学や遺伝学でますます重要になっている。我々の方法は、形質と体サイズの全範囲にわたって形態学的スケーリングの関係の推定を可能にします。形質のサイズが本体サイズ（例えば、13,14）に直線的に拡張できない場合があるので、これは、重要になる可能性があります。動物が唯一の理想的な実験室の条件下で飼育されている場合は、これは明らかにできない場合があります。 我々のプロトコルがショウジョウバエにおける翼胴サイズのスケーリングの関係の推定を可能にするために明示的に開発されたが、それはハエの任意の形態学的特性のスケーリング関係を推定する形質固有の変更で使用することができます。同様に、食事の操作は、おそらく他の完全に変成昆虫の形質と体サイズの全範囲を生成するために使用することができます。このようなアプリケーションは、単にMVW Eを達成し、個体発生でその時点で操作を適用しているときに知る必要があります。

experimental manipulation of body size to estimate morphological scaling relationships in drosophila

体の部分のスケーリングは、動物の形態1-7の中心的な機能です。種内では、形態学的形質が正しく機能する生物のために身体に比例する必要がある、大規模な個人は、通常、大規模な体の部分と小さな個人の一般的に小さい体の部分を持っている、ように全体的なボディシェイプは、成人の体の大きさの範囲にわたって維持されます。正しい割合の要件は、種内の個体は、通常、相対的な特性の大きさで低いばらつきを示すことを意味します。対照的に、相対的な特性の大きさは、種間で劇的に変化し、形態的多様性が生産される主要なメカニズムであることができる。比較作業の世紀にわたって3,4これらの内と種間のパターンを確立しています。 おそらく、この変化を記述するために最も広く使用されているアプローチは、y = xとyがそのような臓器や体の大きさ8として、2つの特徴のサイズがbxα、アロメトリー式を使用して、2つの形態形質の大きさとの間のスケーリングの関係を計算することです、9。両方のサイズが変わるように、この方程式は、2つの特徴間のグループ内（例えば、種、個体群）のスケーリングの関係を説明します。この式の対数変換は単純な線形方程式を生成する、ログは（Y）=ログ（B）+αlog（x）と同じ種の個体間で異なる形質の大きさの対数プロットは、通常、切片を持つ線形スケーリングを明らかにログ（B）とαの傾きから、&quot;相対成長係数&quot;9,10と呼ばれる。群間の形態学的変化は、特定の形質のペアのためのスケーリングの関係のインターセプトや傾きの違いによって説明されています。その結果、相対成長式のパラメータの変化（bおよびα）はエレガントな生物学的なグループ内との間で臓器や体の大きさの関係（11,12を参照）で取得された形状の変化を説明します。 すべての形質が互いにまたはそのため体の大きさ（例えば、13,14）が直線的な比例関係ではない、形態学的スケーリングの関係は、データが特徴サイズの全範囲から取られている場合に最も有益です。ここでは、食事の簡単な実験的操作が昆虫の体の大きさの全範囲を生成するために使用できる方法について説明します。これは、サイズおよび生物学的なグループの間でスケーリングの関係のパラメータの堅牢な比較による形状のcovaries方法の完全な記述を可能にする、特性の任意のペアのためのフルスケールの関係の推定を可能にします。我々はショウジョウバエに焦点を当てていますが、私たちの方法論は、ほぼすべての完全に変成昆虫にも適用できるものです。

Watch this Scientific Journal Video about Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila at JoVE.com

Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila

形態学的スケーリングの関係はorganismal形状をキャプチャして説明します。我々は完全に変成昆虫の体の大きさの自然な範囲内で形態学的スケーリングの関係を測定する方法を提示する。簡単なダイエットの操作を使用して、我々は、形状とサイズの共同どのように変化するかの正確な記述を可能にして、形質の大きさの分布を増加させる。

に形態学的スケーリングの関係を推定するためにボディサイズの実験的操作ショウジョウバエ</em

The scaling of body parts is a central feature of animal morphology1-7. Within species, morphological traits need to be correctly proportioned to the body ...

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Biology

Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila

13.7K ビュー.  University of Houston. 形態学的スケーリングの関係はorganismal形状をキャプチャして説明します。我々は完全に変成昆虫の体の大きさの自然な範囲内で形態学的スケーリングの関係を測定する方法を提示する。簡単なダイエットの操作を使用して、我々は、形状とサイズの共同どのように変化するかの正確な記述を可能にして、形質の大きさの分布を増加させる。

ビデオ: Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila

Experimental Approaches to Tissue Engineering

組織工学への実験的アプローチ

生物学

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

出芽酵母のタンパク質濃度の関数としての形態学的表現型の開発の分析

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

リビングでのイメージング、細胞の形状の変更ショウジョウバエ胚

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

線虫における身体サイズ調節に関与する遺伝子をスクリーニングするための戦略を給餌RNA媒介干渉を用いた<em&gt; Cエレガンス</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

アポトーシス細胞のクリアランスのイメージング中に生きる<em&gt;ショウジョウバエ</em&gt;胚

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.
Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. 
Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

A method for isolation of exosomes from whole blood and further analysis by nanoparticle tracking using a semi-automatic instrument is presented in this article. The presented technology provides an extremely sensitive method for visualizing and analyzing particles in liquid suspension.

エキソソームの定量化とサイズの測定のための革新的方法

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about ...

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ecologically-important endpoints, though they are rarely considered in toxicological studies. D. melanogaster is emerging as a toxicology model for the behavioral, neurological, and genetic impacts of toxicants, to name a few. More importantly, species in the genus Drosophila can be utilized as a model system for an integrative framework approach to incorporate emergent properties and answer ecologically-relevant questions in&#160;toxicology research. The aim of this paper is to provide a protocol for exposing species in the genus Drosophila to pollutants to be used as a model system for a range of phenotypic outputs and ecologically-relevant questions. More specifically, this protocol can be used to 1) link multiple biological levels of organization and understand the impact of toxicants on both individual- and population-level fitness; 2) test the impact of toxicants at different stages of developmental exposure; 3) test multigenerational and evolutionary implications of pollutants; and 4) test multiple contaminants and stressors simultaneously.

Experimental Protocol for Using Drosophila As an Invertebrate Model System for Toxicity Testing in the Laboratory

In this paper, we provide a detailed protocol for exposing species in the genus Drosophila to pollutants with the goal of studying the impact of exposure on a range of phenotypic outputs at different developmental stages and for more than one generation.

無脊椎動物モデル システムとしてショウジョウバエを使用して毒性を実験室でテストするための実験プロトコル

Emergent properties and external factors (population-level and ecosystem-level interactions, in particular) play important roles in mediating ...

Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time through noninvasive monitoring of fluorescence emitted by implanted ectopic human endometrial tissue. For this purpose, biopsies of human endometrium are obtained from donor women ongoing oocyte donation. Human endometrial fragments are cultured in the presence of adenoviruses engineered to express cDNA for the reporter fluorescent protein mCherry. Upon visualization, labeled tissues with an optimal rate of fluorescence after infection are subsequently chosen for the implantation in recipient mice. One week prior to the implantation surgery, recipient mice are oophorectomized, and estradiol pellets are placed subcutaneously to sustain the survival and growth of lesions. On the day of surgery mice are anesthetized, and peritoneal cavity accessed through a small (1.5 cm) incision by the linea-alba. Fluorescently labeled implants are tweezed, briefly soaked in glue and attached to the peritoneal layer. Incisions are sutured, and animals left to recover for a couple of days. Fluorescence emitted by endometriotic implants is usually non-invasively monitored every 3 days for 4 weeks with an in vivo imaging system. Variations in the size of endometriotic implants can be estimated in real time by quantification of the mCherry signal and normalization against the initial time-point showing maximal fluorescence intensity.
Traditional preclinical rodents of models of endometriosis do not allow non-invasive monitoring of lesion in real time but rather allow evaluation of the effects of drugs assayed at the end point. This protocol allows one to track lesions in real time and is more useful to explore the therapeutic potential of drugs in preclinical models of endometriosis. The main limitation of the model thus generated is that non-invasive monitoring is not possible over long periods of time due to the episomal expression of Ad-virus.

Here, we present a protocol for live-imaging of fluorescently labeled human endometrial fragments grafted in mice. The method allows studying the effects of drugs of choice on endometriotic lesion size through monitoring and quantification of fluorescence emitted by the fluorescent reporter on real time

子宮内膜症の異種マウスモデルで病巣の大きさの非侵襲的モニタリング

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time ...

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

ポルシン卵母細胞カプセル化のためのタンパク質分解アルギン酸ヒドロゲルおよび疎水性マイクロバイオリアクター

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the executor for most of the liver functions, primary hepatocytes have limited proliferating capacity. This requires the establishment of ex vivo hepatocyte expansion models for liver physiological and pathological research. Here, we isolated murine hepatocytes by two steps of collagenase perfusion and established a 3D organoid culture as the 'mini-liver' to recapitulate cell-cell interactions and physical functions. The organoids consist of heterogeneous cell populations including progenitors and mature hepatocytes. We introduce the process in detailed to isolate and culture the murine hepatocytes or fetal hepatocyte to form organoids within 2-3 weeks and show how to passage them by mechanically pipetting up and down. In addition, we will also introduce how to digest the organoids into single cells for lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering. The organoids can be used for drug screens, disease modelling, and basic liver research by modeling liver biology and pathobiology.

A long-term ex vitro 3D organoid culture system was established from mouse hepatocytes. These organoids can be passaged and genetically manipulated by lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering.