We gratefully thank the DFG for financial support (HO 1779/21-1) as well as Katrin Kumke and Dr. Veit Schubert (IPK, Gatersleben) for technical advice.

1.染色体の準備<ol><li>種子の発芽や根の先端の固定<ol><li> 22〜24℃で2日間暗条件下でのペトリ皿中の湿ったろ紙の二つの層の10〜20大麦種子を発芽。カミソリの刃を使用することにより、種子中1〜センチ長さと活発な根を切り取ります。 </li><li>砕いた氷 - 水に冷たい水道水を入れた500mlのガラス製ボトルを置くことにより、氷のように冷たい水を準備します。氷のように冷たい水を曝気し、中期細胞の頻度を増加させるために20時間のルートヒントを浸します。 </li><li>酢酸（3：1）を室温で2日間それらを修正するための固定液50mlのエタノールに水から根を転送します。酢酸（3：1）年まで使用まで4℃で固定液新たに調製されたエタノールに根を保管してください。 </li></ol></li><li>洗浄および酵素処理<ol><li> 50mlのガラスビーカーを使用して二回5分間、30ミリリットルの氷冷水道水で10〜20根を洗ってください。双眼顕微鏡を使用してください。 1によって根の1を転送鉗子を用いて、30 mlの0.01 Mクエン酸緩衝液（0.01 Mクエン酸+ 0.01 Mクエン酸ナトリウム、pHは4.8）と二回5分間、ガラス製ビーカーを振盪することにより洗浄します。完全に液体を除去し、カットオフ望ましくない非分裂組織をカミソリの刃を使用して濾紙上に根を置きます。 </li><li>時計皿で植物組織を柔らかくするために約50分（ 表1）37℃で1 mlの酵素混合物中の20根の先端までインキュベートします。酵素混合物は、0.01 Mクエン酸緩衝液で希釈した0.7％のセルラーゼR10、0.7％セルラーゼ、1％のペクトリアーゼと1％のcytohelicaseが含まれています。 -20℃の酵素混合物を保管し、5回まで再利用されます。 </li><li>ピペッティングにより酵素を除去し、残存酵素を置き換えるために二回5ミリリットルの0.01Mクエン酸緩衝液で氷の上のルートヒントを洗います。 </li></ol></li><li>ルートマセラシオン<ol><li> 1ミリリットル同じ時計皿で二回慎重に96％エタノールで洗浄根端。新たに調製した固定液（25％エタノール、75％酢酸）でエタノールを交換してください。 10〜15を使用してください根端あたりμlの固定液。 </li><li>解剖針やピンセットで2 mlチューブに固定剤と一緒に転送ルートヒントや崩壊ルート分裂組織。細胞懸濁液を得るために、細胞を再懸濁するためにチューブを20回タップします。 2ヶ月のCまで-20℃で細胞懸濁液を保管してください。 </li></ol></li><li>細胞懸濁液の滴下<ol><li> 50℃のホットプレート上で水に浸したティッシュペーパーの2-3層を配置します。 30分間冷蔵庫で氷のように冷たい水道水に顕微鏡スライドを浸し。そして、湿ったティッシュペーパーの上にスライドを配置。 </li><li>細胞懸濁液のピペット7-10μL、ホットプレート上に載せ冷却スライド上に20cmの距離からそれをドロップします。ピペットスライド上の細胞懸濁液と同じ場所に酢酸 - エタノール混合物の10μlの、さらに2分間ホットプレート上でスライドを維持します。ウェットティッシュなしでホットプレート上でスライドを置き、1分間乾燥してみましょう。 </li></ol></li><li>品質管理とstoragスライドの電子<ol><li>染色体スプレッドの品質を制御するために位相差顕微鏡を用いてスライドをチェックします。 -20℃のコプリンジャーに96％エタノールに浸漬することにより、同じ日またはストアのいずれかのスライドを使用してください。 </li></ol></li><li> FISH前スライドの前処理;全ての工程は室温で実施されます<ol><li> 5分間の2×SSCの50ミリリットルを含むコプリンジャーに入れスライド（20×SSCは、3M NaClおよび300 mMのクエン酸三ナトリウムが含まれています）。ピンセットを使用して、転送は、3-10分間、45％酢酸50 mlを含むコプリンジャーにスライドします。 </li><li>転送は、10分間の2×SSCの50 mlを含むコプリンジャーにスライドします。転送が（2×SSC中）4％ホルムアルデヒドの50ミリリットルを含むコプリンジャーにスライドし染色体を修正するために10分間スライドを浸します。 </li><li> 50ミリリットルの2×SSCを含むコプリンジャーに、4分間ずつスライドを3回すすぐことにより、ホルムアルデヒドを除去します。それぞれ、70％、90％及び100％エタノールシリーズ中で2分間、コプリンジャーにスライドを脱水し、垂直で乾燥したスライド位置。 </li></ol></li></ol> in situハイブリダイゼーション2.蛍光（FISH） <ol><li>各スライドには、脱イオン化ホルムアミド10μlの、4倍のハイブリダイゼーションバッファーを5μl（200μlのバッファーは80μlの20倍のSSCが含まれ、8μlの1 Mトリス塩酸pH8.0の、1.6μlの0.5 Mを使用して、合計で20μLのハイブリダイゼーション溶液を調製EDTA、11.2μLを10μg/μlのサケ精子と99.2μlのDNaseを含まない水）、プローブの3μLおよびDNアーゼを含まない水を2μl。 </li><li>スライドごとに、ハイブリダイゼーション溶液20μlを加え、24×32ミリメートルのカバースリップで覆い、ゴムセメントでカバースリップを逮捕。ホットプレート上で2分間80℃で同時にプローブを用いてスライドを変性させます。 </li><li>転送は、湿ったチャンバーにスライドして光を避け、37°のCO / Nでスライドをインキュベートします。 2×SSCでコプリンジャーにスライドを洗浄することによってカバースリップを外します。 55〜60℃、2×SSCを含むコプリンジャーにスライドを置き、20分間インキュベート。 </li><li>室温で2分間、コプリンジャーにSSCを2倍のスライドを置きます。それぞれ、70％、90％及び100％エタノールシリーズ中で2分間、コプリンジャーにスライドを脱水します。 </li><li> 、抗フェードマウンティング培地中1μg/ mlの4 &#39;、6-ジアミジノ-2- phenylindoline（DAPI）とスライドと対比を空気乾燥に強い光を避けます。 </li></ol> 3.顕微鏡分析およびストレージ<ol><li>落射蛍光顕微鏡を用いてスライドを分析します。フィルタの選択は、プローブ標識のために使用される蛍光色素に依存します。必要に応じて、年までの暗条件下で4℃で保存するスライド。 </li></ol>

<ol>
	<li>Geber, G., Schweizer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochemical+heterochromatin+differentiation+in+Sinapis+alba+(Cruciferae)+using+a+simple+air-drying+technique+for+producing+chromosome+spreads.">Cytochemical heterochromatin differentiation in Sinapis alba (Cruciferae) using a simple air-drying technique for producing chromosome spreads.</a> Pl Syst Evol. 158 (2-4), 97-106 (1988).</li><li>Andras, S. 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=A+drop-spreading+technique+to+produce+cytoplasm-free+mitotic+preparations+from+plants+with+small+chromosomes.">A drop-spreading technique to produce cytoplasm-free mitotic preparations from plants with small chromosomes.</a> Chromosome Res. 7 (8), 641-647 (1999).</li><li>Rothfels, K. H., Siminovitch, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+air-drying+technique+for+flattening+chromosomes+in+mammalian+cells+grown+in+vitro.">An air-drying technique for flattening chromosomes in mammalian cells grown in vitro.</a> Stain Technology. 33 (2), 73-77 (1958).</li><li>Martin, R., Busch, W., Herrmann, R. G., Wanner, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+preparation+of+plant+chromosomes+for+high-resolution+scanning+electron+microscopy.">Efficient preparation of plant chromosomes for high-resolution scanning electron microscopy.</a> Chromosome Res. 2 (5), 411-415 (1994).</li><li>Kato, A., Albert, P. S., Vega, J. M., Birchler, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+fluorescence+in+situ+hybridization+signal+detection+in+maize+using+directly+labeled+probes+produced+by+high+concentration+DNA+polymerase+nick+translation.">Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation.</a> Biotech. Histochem. 81 (2-3), 71-78 (2006).</li><li>Kirov, I., Divashuk, M., Van Laere, K., Soloviev, A., Khrustaleva, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+easy+'SteamDrop'+method+for+high+quality+plant+chromosome+preparation.">An easy 'SteamDrop' method for high quality plant chromosome preparation.</a> Mol. Cytogenet. 7, 21 (2014).</li><li>Spurbeck, J. L., Zinsmeister, A. R., Meyer, K. J., Jalal, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+chromosome+spreading.">Dynamics of chromosome spreading.</a> Am J Med Genet. 61 (4), 387-393 (1996).</li><li>Ma, 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=Synteny+between+Brachypodium+distachyon+and+Hordeum+vulgare+as+revealed+by+FISH.">Synteny between Brachypodium distachyon and Hordeum vulgare as revealed by FISH.</a> Chromosome Res. 18 (7), 841-850 (2010).</li><li>Kato, A., Lamb, J. C., Birchler, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+painting+using+repetitive+DNA+sequences+as+probes+for+somatic+chromosome+identification+in+maize.">Chromosome painting using repetitive DNA sequences as probes for somatic chromosome identification in maize.</a> Proc. Natl. Acad. Sci. U S A. 101 (37), 13554-13559 (2004).</li><li>Pan, W. H., Houben, A., Schlegel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+effective+cell+synchronization+in+plant-roots+by+hydroxyurea+and+amiprophos-methyl+or+colchicine.">Highly effective cell synchronization in plant-roots by hydroxyurea and amiprophos-methyl or colchicine.</a> Genome. 36 (2), 387-390 (1993).</li><li>Kim, J. S., 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=Integrated+karyotyping+of+sorghum+by+in+situ+hybridization+of+landed+BACs.">Integrated karyotyping of sorghum by in situ hybridization of landed BACs.</a> Genome. 45 (2), 402-412 (2002).</li><li>Lapitan, N. L. V., Brown, S. E., Kennard, W., Stephens, J. L., Knudson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FISH+physical+mapping+with+barley+BAC+clones.">FISH physical mapping with barley BAC clones.</a> Plant J. 11 (1), 149-156 (1997).</li><li>Aliyeva-Schnorr, 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=Cytogenetic+mapping+with+centromeric+BAC+contigs+shows+that+this+recombination-poor+region+comprises+more+than+half+of+barley+chromosome+3H.">Cytogenetic mapping with centromeric BAC contigs shows that this recombination-poor region comprises more than half of barley chromosome 3H.</a> Plant J. 84, 385-394 (2015).</li></ol>

The authors have nothing to disclose.

染色体の調製実験は、イネ科（イネ科）に属している穀物の若い根を使用して行われています。すべての分析種は、二倍体ゲノムセットで14比較的長い分裂中期染色体（11-15μm）を持っており、大規模なゲノム種（5.1から7.9ポンド）に帰属します。 発芽根の長さは、分裂組織の最大値を得るために、2cm以上ではなかったです。分裂細胞の同期化は、分裂中期の量が10を...

蛍光in situハイブリダイゼーション（FISH）は、染色体レベルでのシングル、高コピー配列の物理的マッピングのための効果的なツールです。前提条件は、高品質の染色体スプレッドの調製です。動物および植物細胞にも同様に適しているであろう全く一般的な染色体調製プロトコルはありません。植物染色体の調製により堅い細胞壁と異なる種内の様々な細胞質の一貫性に特に困難です。植物染色体の調製のための有利な方法の一つは、また、ドロップ拡散技術、空気乾燥技術1,2として知られる、いわゆるドロップ技術です。この方法は、まずin vitroで増殖させた哺乳動物細胞の3のためロートフェルスとSiminovitchによって1958年に導入されました。その後、マーティンら 4と ​​加藤ら 5は、植物のため、この方法を適応しました。 最近では、メソッドの名前」SteamDrop＆＃39;非重複染色体6の製造のための水の蒸気を使用する開発されました。 、高湿度の正の影響が早く7観察されたが、「SteamDropは「高品質の染色体標本6の制御されたワークフローを提供します。蒸気処理は、おそらく染色体タンパク質のいくつかの修正に接続染色体の伸びが発生します。完全な中期の十分な数の保持、その後のFISH実験に広がるものの、中期スプレッドを結果の品質が非常に高く、技術的な専門知識を必要とします。 ここでは、単一および高コピープローブ5,8のFISH検出に適し分裂穀物染色体の調製のためのプロトコルを提示します。この方法は、加藤50％-55％の相対湿度下で行わ9（ 図1）によって記載され空気乾燥滴下法の改良された変異体です。このプロトコルは、削減された数を含み、そのアプリケーションは、簡単に、効率的かつ再現可能な行う洗浄工程の。このプロトコルを用いて、我々は、 オオムギ、Hのbulbosum、Hのmarinumの、Hのmurinum、Hのpubiflorumとライムギのための高品質の染色体スプレッドおよびFISHの結果を得ました。

<table>
	<tbody>
		<tr>
			<td>Hot Plate</td>
			<td>MEDAX GmbH</td>
			<td>12603</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulase R10</td>
			<td>Duchefa</td>
			<td>C8001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cellulase&#160;</td>
			<td>CalBioChem</td>
			<td>219466</td>
			<td></td>
		</tr>
		<tr>
			<td>Pectolyase</td>
			<td>Sigma</td>
			<td>P3026</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytohelicase</td>
			<td>Sigma</td>
			<td>C8274</td>
			<td></td>
		</tr>
		<tr>
			<td>Texas Red-12-dUTP</td>
			<td>Invitrogen</td>
			<td>C3176</td>
			<td>direct fluorochrome&#160;</td>
		</tr>
		<tr>
			<td>Fluor488-5-dUTP</td>
			<td>Invitrogen</td>
			<td>C11397</td>
			<td>direct fluorochrome&#160;</td>
		</tr>
		<tr>
			<td>Fluorecsence microscope</td>
			<td>Olympus BX61</td>
			<td>BX61</td>
			<td></td>
		</tr>
		<tr>
			<td>CCD camera</td>
			<td>Orca ER, Hamamatsu</td>
			<td>C10600</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8217;,6-diamidino-2-phenylindole (DAPI)&#160;</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td>fluorecsent dye</td>
		</tr>
	</tbody>
</table>

a fast air-dry dropping chromosome preparation method suitable for fish in plants

染色体スプレッドの調製は、 蛍光in situハイブリダイゼーション（FISH）の成功した性能のための前提条件です。高品質の植物染色体スプレッドの調製により堅い細胞壁に困難です。植物染色体の調製のための承認された方法の一つは、いわゆるドロップ製剤、またドロップ拡散または空気乾燥技術として知られています。ここでは、有糸分裂染色体の高速製造のためのプロトコルは、シングルおよび高コピーDNAプローブのFISH検出に適し広がる提示します。この方法では、50％-55％の相対湿度下で行わ空気乾燥ドロップ方式の改良された変異体です。このプロトコルは、そのアプリケーションは、簡単に、効率的かつ再現可能な行う洗浄工程の数の減少を含みます。このアプローチの明白な利点は、成功したFISH分析に最適な前提条件となる損傷を受けていないと、多数の中期染色体よく普及しています。このプロトコルを用いて、我々は、高品質のCHROMを取得しましたオオムギ、Hのbulbosum、Hのmarinumの、Hのmurinum、Hのpubiflorumとライムギのためosomeスプレッドと再現性のFISH結果。

分裂中期スプレッドと顕微鏡スライドは、上記高速の空気乾燥滴下染色体の調製方法により調製した（ 補遺図1）。 FISH分析は、両方の反復及び単一コピー配列を用いて行きました。画像は、対応するフルオロフォアの励起を可能にするフィルタのセットを用いて落射蛍光顕微鏡によって得られた、高感度CCDモノクロカメラで撮影しました。画像取得のために、我々は、画像取得?...

Watch this Scientific Journal Video about A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants at JoVE.com

A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

植物におけるFISHのための適切な高速空気乾燥滴下染色体製造方法

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

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Research

JoVE Journal

Biology

13.1K ビュー.  Leibniz Institute of Plant Genetics and Crop Plant Research (IPK). A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

ビデオ: A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

パイロシーケンシング：正確なジェノタイピングのための簡単​​な方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

生物学

Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological advantages provided by the giant salivary gland polytene chromosomes of third instar larvae.  In this tissue the chromosomes undergo many rounds of replication in the absence of cell division giving rise to approximately 1000 copies.  The DNA remains aligned after each replicative cycle resulting in greatly enlarged chromosomes that provide a unique opportunity to correlate chromatin morphology with the localization of specific proteins.  Consequently, there has been a high level of interest in defining the epigenetic modifications present at different genes and at different stages of the transcription process. An important tool for such studies is the labeling of polytene chromosomes with antibodies to the enzyme, transcription factor, or histone modification of interest. This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

This video protocol illustrates the squash technique used in the Johansen laboratory to prepare Drosophila polytene chromosomes for antibody labeling.

抗体標識用ショウジョウバエ多糸染色体のカボチャの準備

Drosophila has long been a favorite model system for studying the relationship between chromatin structure and gene regulation due to the cytological ...

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies1,2. Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell3-5, but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography6,7. These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications.

An ex-ovo Chicken Embryo Culture System Suitable for Imaging and Microsurgery Applications

In this article, we present a simple methodology to enable long-term ex-ovo avian embryo culture. This technique is ideal for longitudinal experimentation requiring complete optical accessibility and/or sterile transportation in avian embryos.

アン EX - OVO</emイメージングとマイクロサーアプリケーションに適して>ニワトリ胚培養システム

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

アイスキャップ：成長させる方法シロイヌナズナ</emハイスループットジェノタイピング用96ウェルプレートで>とトマト

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Chromosome Preparation From Cultured Cells

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as fluorescence in situ hybridization (FISH), this assay has the powerful ability to analyze individual cells for aberrations that involve gains or losses of portions of the genome&#160;and rearrangements involving one or more chromosomes. In humans, chromosome abnormalities occur in approximately 1 per 160 live births1,2, 60-80% of all miscarriages3,4, 10% of stillbirths2,5, 13% of individuals with congenital heart disease6, 3-6% of infertility cases2, and in many patients with developmental delay&#160;and birth defects7. Cytogenetic analysis of malignancy is routinely used by researchers and clinicians, as observations of clonal chromosomal abnormalities have been shown to have both diagnostic and prognostic significance8,9.&#160; Chromosome isolation is invaluable for gene therapy and stem cell research of organisms including nonhuman primates and rodents10-13.
Chromosomes can be isolated from cells of live tissues, including blood lymphocytes, skin fibroblasts, amniocytes, placenta, bone marrow, and tumor specimens. Chromosomes are analyzed at the metaphase stage of mitosis, when they are most condensed and therefore more clearly visible. The first step of the chromosome isolation technique involves the disruption of the spindle fibers by incubation with Colcemid, to prevent the cells from proceeding to the subsequent anaphase stage. The cells are then treated with a hypotonic solution and preserved in their swollen state with Carnoy&#39;s fixative. The cells are then dropped on to slides and can then be utilized for a variety of procedures. G-banding involves trypsin treatment followed by staining with Giemsa to create characteristic light and dark bands. The same procedure to isolate chromosomes can be used for the preparation of cells for procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY)14,15.

Chromosomes can be isolated from live cells such as lymphocytes or skin fibroblasts, and from organisms including humans or mice. These chromosome preparations can be further utilized for routine G-banding and molecular cytogenetic procedures such as fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH), and spectral karyotyping (SKY).

培養細胞からの染色体の準備

Chromosome (cytogenetic) analysis is widely used for the detection of chromosome instability. When followed by G-banding and molecular techniques such as ...

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 ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

組み合わせたDNA-RNA蛍光<em&gt;その場で</em&gt;ハイブリダイゼーション（FISH）差別雌マウス胚性幹細胞におけるX染色体の不活性化を研究するために、

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Preparation of Rat Serum Suitable for Mammalian Whole Embryo Culture

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for investigating the mechanisms of developmental processes. Immediately centrifuged (IC) rat serum is commonly used for WEC and is essential for the growth and development of cultured mouse and rat embryos ex vivo. For the culture of midgestation embryos (i.e., E8.0-12.5 for the mouse, and E10.0-14.5 for the rat), 100% rat serum is the best media for supporting the growth of the embryo ex vivo. To prepare rat serum suitable for WEC, the collected blood should be centrifuged immediately to separate the blood cells from the plasma fraction. After centrifugation, the fibrin clot forms in the upper layer; this clot should be squeezed gently using a pair of sterile forceps and subsequently centrifuged to completely separate the blood cells from the serum. In this video article, we demonstrate our standard protocol for the preparation of optimal IC rat serum, including blood collection from the abdominal aorta of male rats and extraction of the serum by centrifugation.

Mammalian whole embryo culture (WEC) is widely used in teratology and developmental biology. Immediately centrifuged rat serum is commonly provided as a medium for both mouse and rat WEC. In this video, we demonstrate our standard protocol for the preparation of high-quality rat serum suitable for mammalian WEC.

哺乳類の全胚培養に適したラット血清の調製

Mammalian whole embryo culture (WEC) is a widely used technique for examining pharmacological toxicity in developing mouse and rat embryos and for ...

Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on the surface of slides (rather than dropping them from a height), we demonstrate an optimized technique on mouse spermatocytes that was first described in 1997. This method is widely used in laboratories to study mammalian meiosis because it yields a plethora of high quality nuclei undergoing substages of prophase I. Seminiferous tubules are first placed in a hypotonic solution to swell spermatocytes. Then spermatocytes are released into a sucrose solution to create a cell suspension, and nuclei are spread onto fixative-soaked glass slides. Following immunostaining, a diversity of proteins germane to meiotic processes can be examined. For example, proteins of the synaptonemal complex, a tripartite structure that connects the chromosome axes/cores of homologs together can be easily visualized. Meiotic recombination proteins, which are involved in repair of DNA double-strand breaks by homologous recombination, can also be immunostained to evaluate progression of prophase I. Here we describe and demonstrate in detail a technique widely used to study mammalian meiosis in spermatocytes from juvenile or adult male mice.

Meiosis is the developmental process by which gametes are formed through a single round of DNA replication and two successive rounds of chromosome segregation. Mammalian meiosis can be examined by utilizing a technique to prepare meiotic chromosome spreads. Here, we demonstrate a method of preparing surface-spread nuclei from mouse spermatocytes.

マウス精母細胞から減数分裂期染色体の準備をスプレッドします。

Mammalian meiosis is a dynamic developmental process that occurs in germ cells and can be studied and characterized. Using a method to spread nuclei on ...

Standardizing a Non-Lethal Method for Characterizing the Reproductive Status and Larval Development of Freshwater Mussels (Bivalvia: Unionida)

Actively monitoring the timing, development, and reproductive patterns of endangered species is critical when managing for population recovery. Freshwater mussels are among the most imperiled organisms in the world, but information about early larval (glochidial) development and brooding periods is still lacking for many species. Previous studies have focused on the complex life history stage when female mussels are ready to parasitize host fish, but few studies have focused on the brooding period and timing of larval development. The protocol described here allows researchers to non-lethally evaluate the state of gravidity for female mussels. The results of this study show that this method does not affect a female mussel&rsquo;s ability to stay gravid or become gravid again after sampling has been performed. The advantage of this method may permit its use on federally threatened or endangered species or other populations of high conservation concern. This protocol can be adapted for use on both preserved or live individuals and was tested on a variety of mussel species. The database provided is a repository for a breadth of information on timing of reproductive habits and will facilitate future freshwater mussel research, conservation, and recovery efforts.

Standardizing a Non-Lethal Method for Characterizing the Reproductive Status and Larval Development of Freshwater Mussels Bivalvia: Unionida

Freshwater mussel conservation is dependent on monitoring reproductive patterns and processes of species. This study standardizes a non-lethal protocol for sampling gill contents, characterizing larval development, and providing a digital repository for data collected. This protocol-database package will be an important tool for mussel researchers in recovery of imperiled species.

淡水ムール貝の生殖状態と幼虫発達を特徴付けるための非致死的方法の標準化(ビバルビア:ユニオンダ)

Actively monitoring the timing, development, and reproductive patterns of endangered species is critical when managing for population recovery. Freshwater ...