Name: a fast air-dry dropping chromosome preparation method suitable for fish in plants
Uploaded: 2015-12-16T14:00:00.000+00:00
Duration: 9 min 8 s
Description: Watch this Scientific Journal Video about A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants at JoVE.com

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>
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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. 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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&amp;nbsp;</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&amp;nbsp;</td></tr><tr><td>Fluor488-5-dUTP</td><td>Invitrogen</td><td>C11397</td><td>direct fluorochrome&amp;nbsp;</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&amp;rsquo;,6-diamidino-2-phenylindole (DAPI)&amp;nbsp;</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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Biology

13.2K 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

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

In Vivoモデル システムとしてゼブラフィッシュを用いた DNA 複製タイミングのプロファイリング

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

遺伝学

High-throughput DNA Extraction and Genotyping of 3dpf Zebrafish Larvae by Fin Clipping

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short generation time, are easy to handle, display a high reproductive rate, low cost, and are easily amenable to genetic manipulations by microinjection of DNA in embryos. Recent advances in gene editing tools are enabling precise introduction of mutations and transgenes in zebrafish. Disease modeling in zebrafish often leads to larval phenotypes and early death which can be challenging to interpret if genotypes are unknown. This early identification of genotypes is also needed in experiments requiring sample pooling, such as in gene expression or mass spectrometry studies. However, extensive genotypic screening is limited by traditional methods, which in most labs are performed only on adult zebrafish or in postmortem larvae. We addressed this problem by adapting a method for the isolation of PCR-ready genomic DNA from live zebrafish larvae that can be achieved as early as 72 h post-fertilization (hpf). This time and cost-effective technique, improved from a previously published genotyping protocol, allows the identification of genotypes from microscopic fin biopsies. The fins quickly regenerate as the larvae develop. Researchers are then able to select and raise the desired genotypes to adulthood by utilizing this high-throughput PCR-based genotyping procedure.

Zebrafish have been used as reliable genetic model organisms in biomedical research, especially with the advent of gene-editing technologies. When larval phenotypes are expected, DNA extraction and genotype identification can be challenging. Here, we describe an efficient genotyping procedure for zebrafish larvae, by tail clipping, as early as 72-h post-fertilization.

ハイスループット DNA の抽出およびフィン クリッピングが 3dpf ゼブラフィッシュ仔のジェノタイピング

Zebrafish (Danio rerio) possess orthologues for 84% of the genes known to be associated with human diseases. In addition, these animals have a short ...

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

熱ショック2処理とゼブラフィッシュ胚の倍数性マニピュレーション

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

発生生物学

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

母親の遺伝子産物の機能操作の使用<em&gt;インビトロ</emゼブラフィッシュにおける&gt;卵成熟

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

蛍光灯<em&gt;その場で</em蚊の分裂期染色体上&gt;ハイブリダイゼーション

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

免疫・感染学

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

ゼブラフィッシュの精子細胞からのマイオサイト染色体の広がりの調製

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding ...

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

の調製ショウジョウバエ</emハンギングドロッププロトコルを使用してライブイメージングのための>胚

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

生物学

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

植物の効率的な方法で複数のキメラ蛍光融合タンパク質の共発現

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

化学

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

ゼブラフィッシュ精子の凍結保存用ハイスループット法とインビトロ受精

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

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

要約

さらに動画を探す

この動画の章

In Vivoモデルシステムとしてゼブラフィッシュを用いた DNA 複製タイミングのプロファイリング

ハイスループット DNA の抽出およびフィンクリッピングが 3dpf ゼブラフィッシュ仔のジェノタイピング

熱ショック2処理とゼブラフィッシュ胚の倍数性マニピュレーション

母親の遺伝子産物の機能操作の使用

蛍光灯

ゼブラフィッシュの精子細胞からのマイオサイト染色体の広がりの調製

の調製ショウジョウバエ胚

植物の効率的な方法で複数のキメラ蛍光融合タンパク質の共発現

ゼブラフィッシュ精子の凍結保存用ハイスループット法とインビトロ受精

火花のサイレンシング:弱い電気魚のCRISPR/Cas9ゲノム編集

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

要約

さらに動画を探す

この動画の章

In Vivoモデル システムとしてゼブラフィッシュを用いた DNA 複製タイミングのプロファイリング

ハイスループット DNA の抽出およびフィン クリッピングが 3dpf ゼブラフィッシュ仔のジェノタイピング

熱ショック2処理とゼブラフィッシュ胚の倍数性マニピュレーション

母親の遺伝子産物の機能操作の使用

蛍光灯

ゼブラフィッシュの精子細胞からのマイオサイト染色体の広がりの調製

の調製ショウジョウバエ胚

植物の効率的な方法で複数のキメラ蛍光融合タンパク質の共発現

ゼブラフィッシュ精子の凍結保存用ハイスループット法とインビトロ受精

火花のサイレンシング:弱い電気魚のCRISPR/Cas9ゲノム編集

In Vivoモデルシステムとしてゼブラフィッシュを用いた DNA 複製タイミングのプロファイリング

ハイスループット DNA の抽出およびフィンクリッピングが 3dpf ゼブラフィッシュ仔のジェノタイピング