私たちは、助言や技術支援のためのBlaschke、メイ、そしてウィトワーラボのメンバーに感謝。この作品は、JLBに、PCMC財団、NIH R01 MH092256とDP2 MH100008、そしてダイム財団研究助成金＃1-FY13-425の3月までサポートされています。

1。 DNAの準備<ol><li> 50mMのKCl、10mMのトリス-HCl、pH8.3、0.3％のTween20、0.3％NP40：DNA溶解緩衝液を調製する。使用12日の1ミリグラム/ mlの最終濃度になるように、新鮮なプロテイナーゼKを追加します。 </li><li>組織収集： <ol><li>成魚フィンクリップの場合： <ol><li>魚を麻酔：0.004％、MS-222（トリカイン）溶液中に魚を置きます。鰓の動きが遅くなるまで待ってください。 </li><li> 5月10日キムワイプのスタック上に魚を入れて滅菌したカミソリの刃で、テールフィン、2〜3mm程度の小片をカット。 </li><li>すぐに回復のための新鮮な水とラベルされたタンクに魚を置き、慎重にフィンクリップが含まれていますタンクとそれに対応するチューブの両方にラベルを付ける。水を変更し、一日おきに魚を食べます。 </li><li>無菌ピペットチップでフィンのクリップをピックアップし、100μlのDNA溶解緩衝液で満たされたチューブに転送します。 </li><li>ピペットチップを廃棄して、鋭利物容器にカミソリの刃を捨てる。 </li></ol></li><li>胚テールクリップの場合： <ol><li> 0.004％のMS-222（トリカイン）溶液中に胚を置きます。 2分待ちます。 </li><li>解剖顕微鏡下でピンセットで尾の部分をカット。 </li><li> 30μlのDNA溶解緩衝液で満たされたラベルチューブに尾を置く。 </li><li>すぐに100μlの4％パラホルムアルデヒドを含むチューブに胚を置く。 </li><li>胚およびそれに対応する尾管をラベルチューブ。 </li><li>固定用の4℃で一晩胚を試験管に保管してください。 </li><li>汚染を最小限に抑えるために各胚の間でミリQ水とキムワイプを使用して鉗子を清掃してください。 </li></ol></li><li>全体の胚の場合： <ol><li> 0.004％のMS-222（トリカイン）溶液中に胚を置きます。 2分待ちます。 </li><li> 100μlのDNA溶解緩衝液で満たされたチューブ内で1-5胚を置く。 Dechorionationは必要ありません。 </li></ol></li></ol></li><li> DNA消化 55℃で組織（FINあるいはテールクリップや胚）でチューブをインキュベートします一晩12に4時間のアップのため。 </li><li>プロテイナーゼKを不活性化 15分12 95℃まで熱管。 DNAは直ちにPCRに使用するか、最大3ヶ月間-20℃で保存する必要があります。 </li></ol> 2。 PCR法<ol><li>設計プライマー手動またはプライマー設計ソフトウェア（ 例えば Lightscannerプライマー設計ソフトウェアBiofire）による。 HRMAのためのPCR産物は、通常、50〜200塩基対であり、SNP検出のためのPCR産物は、典型的には50〜80塩基対、小さくなければならない。 </li><li> PCR法<ol><li> 96ウェルまたは384ウェルプレート中でPCR反応を行う。 </li><li>各プライマー5pmolの、4μlのLightScannerマスターミックス（0.1 UホットスタートTaqポリメラーゼ-ABポリメラーゼ、緩衝液、0.2mMのdNTPを、2mMの塩化マグネシウム、および1×蛍光二本鎖DNA結合色素）：10μlの全容積を使用大人のテールフィンまたは胚の尾13から3μLゲノムDNAから抽出され、1μlのゲノムDNAテンプレート。 </li><li> 30μlのミネラルオイルをカバーサンプル。 </lI&gt; <li>光学的に透明な接着シールでプレートをカバーしています。 </li><li> PCRサイクリング条件は、典型的な最適条件は、95℃で5分および10秒の30サイクル、95℃であり、60°C、72°Cで30秒、25秒、30秒、95℃で終わるに冷却15℃ </li><li>店舗PCRプレート4℃またはHRMAに直接進んでください。 </li><li> PCRの最初の最適化中にアガロースゲル電気泳動を用いてそのサイズを分析することによって我々のPCR産物を確認すると、PCR産物の配列決定により、示されている場合。 </li></ol></li></ol> 3。 HRMA <ol><li>熱板<ol><li>溶融分析システムにプレートを置きます。 </li><li> （LightScannerソフトウェアがコールイット2.0）ソフトウェアを起動します。 </li><li> 60〜95℃から熱板</li><li>新しいデータ·ストレージ·ファイルを作成します。 </li></ol></li><li> HRMAのデータを分析します（ 図2）。 分析の複数のステップが可能です。我々は、データmの最も一般的に使用されるセットを示すanipulations。 <ol><li>サブセットの設定 サブセット]タブをクリックします。サンプルでウェルを選択します。 </li><li>正規化<ol><li>左側のパネルでノーマライズ ] タブを選択します。蛍光分散を解消するために、手動で前（初期）平行二重線を配置し、ポスト（最終）溶融領域（ 図2C、矢頭）示されているように、1から0への全てのサンプルのプリメルトとポスト溶融蛍光シグナルを標準化それぞれ14。 </li><li>溶融領域は前後のメルトライン間の領域になるように線の位置を調整するが、選択されていない。一般的に、低最小と下マックス（アッパー最小アッパー最大）温度ラインは約1℃離して配置する必要があります。 </li><li>すべてのサンプルは、POSに最もよくとして最大値またはプリメルト領域および最小または後の溶融領域に対するいいえ（0％）蛍光（のための完全な（100％）の蛍光を有するように正規化するための温度位置を選択してくださいてアクセスします）。正規化は、融点と0の最小蛍光での溶融点からしてほぼ水平線まで伸びる1の最大蛍光でほぼ水平線になるはずです。1上または0以下の蛍光レベルがあってはならない。たとえば、 図2（c）に、79〜80℃の温度範囲を使用前溶融曲線を正規化</li></ol></li><li>グループ化/クラスタリング それらの融解温度（ 図2C、緑のボックス）に基づいて遺伝子型を区別します。 グループ化を選択します。 <ol><li> グループ化]セクションの下の標準選択リストからAUTOGROUPを選択します。 </li><li>グループセクションの下に感度選択リストから、それぞれの単一の遷移や、複数の遷移にプロファイルを溶融させるための標準または高 ]を選択します。 </li><li> 計算を選択します。グループ化]セクションの下にグループ化します。 </li><li> [ファイル ] メニューに移動し、その結果を保存するために[保存]をクリックします。 </li></ol></li></ol></li></ol>

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著者らは、開示することは何もありません。

HRMAと組み合わせたPCRは、ゼブラフィッシュの遺伝子型解析のための強力な技術です。このアプローチの利点は、その速度、ロバスト性、さらには点突然変異を検出するための感度である。全体のプロトコルは、フィンクリップする溶融曲線解析から、単一の個人によって8時間未満で行うことができる。また、この技術は、ハイスループットスクリーニングのために適している、臭化エチジ?...

ゼブラフィッシュ（ ゼブラフィッシュ ） が広く開発および疾患モデルの研究のために使用脊椎動物モデル系である。最近では、数々のトランスジェニック、突然変異の技術は、ゼブラフィッシュのために開発されている。通常、トランスポゾンシステム1に基づいた迅速な遺伝子導入技術は、複数のDNA断片の集合体2のための改良されたクローン作成オプションと組み合わされている。ジンクフィンガーヌクレアーゼ（ZFNを）および転写活性化因子のようなエフェクターヌクレアーゼ（TALENS）は、ゼブラフィッシュの3,4の両方の体細胞および生殖細胞系列の細胞に遺伝子座を標的とするために使用されてきた。これらの技術は、効率的に高周波突然変異の作成 ​​および生殖系列伝達3,4と、遺伝子改変動物を生成することができる。 これらの進歩にもかかわらず、ゼブラフィッシュの伝統的なジェノタイピング技術は、変異誘発および遺伝子導入ツールのフルパワーを制限している。 PCRは、時にはrestrictioと組み合わせ、ゲル電気泳動、続いてnは消化酵素、広くゲノム改変を検出するために使用されるが、小さな挿入または欠失を同定するための時間がかかり、あまり敏感である。 TaqManプローブアッセイは、高い初期コストが、慎重な最適化を必要とする。 PCR産物の配列決定は数日かかると大規模なスクリーニングには実用的ではないができる。制限断片長多型（RFLP）分析は、唯一の制限酵素認識部位の限られた範囲に影響を与えるSNPを識別することができる。 高解像度融解分析（HRMA）、閉じたチューブのポスト-PCR分析法は、迅速、高感度、安価で、かつ多数のサンプルをスクリーニングに適している最近開発された方法である。 HRMAのSNPは、突然変異、および5-7導入遺伝子を検出するために使用することができる。 HRMAは、二本鎖DNAの熱変性に基づくものであり、各PCRアンプリコンは、特徴的な固有の解離（溶湯）5を有している。サンプルは、tと判別することができるそれらの異なるヌクレオチド組成、GC含量、または長さが、O、通常、二本鎖DNA に結合する8蛍光色素と組み合わせて含む。従って、HRMAは、異なる溶融曲線の特性に基づいて、異なる遺伝子型を区別することができる。 HRMAは、低コストの試薬を使用し、シングルステップのポストPCR処理であるため、ハイスループット戦略のために使用することができる。 HRMAので分析後のPCRアンプリコンが他の用途に使用することができ、非破壊である。 HRMA、細胞株、マウス、およびヒト9-11を含む、多くの生物およびシステムに適用されている。その使用は、最近、ジンクフィンガーヌクレアーゼ（ZFN）およびTALENS 6,12,13によって誘発される突然変異を検出するために、ゼブラフィッシュに記載されている。 本論文では、胚および成体ゼブラフィッシュ（ 図1）でPCRベースHRMAの実行方法について説明します。このプロトコルは、Siを含む、SNPは、導入遺伝子、および突然変異を検出するのに適しているngle塩基対の変化、挿入、または欠失。

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		<tr height="42">
			<td height="42" style="height:42px;width:232px;">100 Reaction LightScanner Master Mix</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;">HRLS-ASY-0002</td>
			<td style="width:157px;">www.biofiredx.com</td>
			<td style="width:167px;">Store at -20 &#176;C&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">Hard-Shell PCR 96-well BLK/WHT Plates</td>
			<td style="width:145px;">Bio-Rad Laboratories</td>
			<td style="width:141px;">HSP9665</td>
			<td>www.bio-rad.com</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Microseal &#39;B&#39; Adhesive Seals</td>
			<td style="width:145px;">Bio-Rad Laboratories</td>
			<td style="width:141px;">MSB1001</td>
			<td>www.bio-rad.com</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">96-Well LightScanner Instrument</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;">LSCN-ASY-0040</td>
			<td>www.biofiredx.com</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">LightScanner Software with Call-IT 2.0</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;"></td>
			<td>www.biofiredx.com</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">High-Resolution Melting Analysis 2.0</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;"></td>
			<td>www.biofiredx.com</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">LightScanner Primer Design Software</td>
			<td style="width:145px;">BioFire</td>
			<td style="width:141px;"></td>
			<td>www.biofiredx.com</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Vector NTI Software</td>
			<td style="width:145px;">Invitrogen</td>
			<td style="width:141px;"></td>
			<td>www.invitrogen.com</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Tricaine</td>
			<td style="width:145px;"></td>
			<td style="width:141px;"></td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Paraformaldehyde</td>
			<td style="width:145px;"></td>
			<td style="width:141px;"></td>
			<td style="width:157px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid and efficient zebrafish genotyping using pcr with high-resolution melt analysis

ゼブラフィッシュは、発達を研究する疾患をモデル化し、薬物スクリーニングを行うための強力な脊椎動物モデル系である。最近、遺伝のさまざまなツールが突然変異を誘発し、トランスジェニック系統を生成するための複数の戦略を含め、導入されている。しかしながら、大規模スクリーニングは、時間がかかり、労働集約的であり、伝統的な遺伝子型決定法によって制限される。ここでは、高分解能融解分析（HRMA）と組み合わせたPCRによるゼブラフィッシュの遺伝子型を分析するための技術が記載されている。このアプローチは、汚染の成果物のリスクが低いと、迅速、高感度、かつ安価である。 HRMAとPCRによる遺伝子型決定は、ハイスループットスクリーニングプロトコルを含めて、胚または成体の魚のために使用することができる。

プロトコルは、1日の間に行うか（作業のフロー図を図1に示されている）、数日間にわたって段階で分離することができる。 DNA抽出は、PCRアンプリコンの溶融および分析が続く。アンプリコンの溶融のための温度は、大きさおよびGC含量に依存するが、一般的に開始し、50˚C 95˚Cの終了温度が（ 図2Aおよび2B）が適切である。溶融が実行されると、蛍光溶?...

Watch this Scientific Journal Video about Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis at JoVE.com

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

高解像度融解分析（HRMA）と合わせPCRは、ゼブラフィッシュの遺伝子型を決定するための迅速かつ効率的な方法として示されている。

高解像度メルト解析を用いたPCRを使用して、迅速かつ効率的なゼブラフィッシュ遺伝子型決定

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

rapid-efficient-zebrafish-genotyping-using-pcr-with-high-resolution

Research

JoVE Journal

Biology

22.1K ビュー.  University of Utah School of Medicine. 高解像度融解分析（HRMA）と合わせPCRは、ゼブラフィッシュの遺伝子型を決定するための迅速かつ効率的な方法として示されている。 

ビデオ: Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

Rapid Genotyping of Mouse Tissue Using Sigma&#039;s Extract-N-Amp Tissue PCR Kit

The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.

シグマのエキス- N - Ampの組織PCRキットを使用してマウス組織の急速なジェノタイピング

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is ...

生物学

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

ゼブラフィッシュの全マウント高分解能二重蛍光上皮内ハイブリダイゼーション

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Rapid PCR Thermocycling using Microscale Thermal Convection

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed1. Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format.
Thermal convection has emerged as a promising alternative thermocycling approach that has the potential to overcome these limitations2-9. Convective flows are an everyday occurrence in a diverse array of settings ranging from the Earth's atmosphere, oceans, and interior, to decorative and colorful lava lamps. Fluid motion is initiated in the same way in each case: a buoyancy driven instability arises when a confined volume of fluid is subjected to a spatial temperature gradient. These same phenomena offer an attractive way to perform PCR thermocycling. By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with the denaturing, annealing, and extension stages of the reaction (Figure 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, completely eliminating the need to repeatedly heat and cool the instrument.
One of the main challenges facing design of convective thermocyclers is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time10,11. Here we describe results of our efforts to probe the full 3-D velocity and temperature distributions in microscale convective thermocyclers12. Unexpectedly, we have discovered a subset of complex flow trajectories that are highly favorable for PCR due to a synergistic combination of (1) continuous exchange among flow paths that provides an enhanced opportunity for reagents to sample the full range of optimal temperature profiles, and (2) increased time spent within the extension temperature zone the rate limiting step of PCR. Extremely rapid DNA amplification times (under 10 min) are achievable in reactors designed to generate these flows.

We describe a novel method to perform DNA replication via the polymerase chain reaction (PCR). Thermal convection is harnessed to continuously shuttle reagents between denaturing, annealing, and extension conditions by maintaining opposing surfaces of the reactor at constant temperature. This inherently simple design promises to make rapid PCR more accessible.

ミクロ熱対流を使用して迅速なPCRの熱サイクル

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

の野生型と観賞用品種を区別するPCRベースのジェノタイピング法チガヤ</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

Using High Resolution Computed Tomography to Visualize the Three Dimensional Structure and Function of Plant Vasculature

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being used to evaluate the structure and function of plant xylem network in three dimensions (3D) (e.g. Brodersen et al. 2010; 2011; 2012a,b). HRCT imaging is based on the same principles as medical CT systems, but a high intensity synchrotron x-ray source results in higher spatial resolution and decreased image acquisition time. Here, we demonstrate in detail how synchrotron-based HRCT (performed at the Advanced Light Source-LBNL Berkeley, CA, USA) in combination with Avizo software (VSG Inc., Burlington, MA, USA) is being used to explore plant xylem in excised tissue and living plants. This new imaging tool allows users to move beyond traditional static, 2D light or electron micrographs and study samples using virtual serial sections in any plane. An infinite number of slices in any orientation can be made on the same sample, a feature that is physically impossible using traditional microscopy methods.
Results demonstrate that HRCT can be applied to both herbaceous and woody plant species, and a range of plant organs (i.e. leaves, petioles, stems, trunks, roots). Figures presented here help demonstrate both a range of representative plant vascular anatomy and the type of detail extracted from HRCT datasets, including scans for coast redwood (Sequoia sempervirens), walnut (Juglans spp.), oak (Quercus spp.), and maple (Acer spp.) tree saplings to sunflowers (Helianthus annuus), grapevines (Vitis spp.), and ferns (Pteridium aquilinum and Woodwardia fimbriata). Excised and dried samples from woody species are easiest to scan and typically yield the best images. However, recent improvements (i.e. more rapid scans and sample stabilization) have made it possible to use this visualization technique on green tissues (e.g. petioles) and in living plants. On occasion some shrinkage of hydrated green plant tissues will cause images to blur and methods to avoid these issues are described. These recent advances with HRCT provide promising new insights into plant vascular function.

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique that can be used to study the structure and function of plant vasculature in 3D. We demonstrate how HRCT facilitates exploration of xylem networks across a wide range of plant tissues and species.

プラントの脈管構造の三次元構造と機能を可視化するために、高分解能CTを用いた

High resolution x-ray computed tomography (HRCT) is a non-destructive diagnostic imaging technique with sub-micron resolution capability that is now being ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

から初期段階の胚の効率的かつ迅速な単離<em&gt;シロイヌナズナ</em&gt;種子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

高解像度全マウント<em&gt;その場で</em遺伝子発現と機能を研究するためにゼブラフィッシュ胚内&gt;ハイブリダイゼーション

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag分析 - 高スループット、Sc2.0ジェノタイピングのためのリアルタイムPCRアッセイ

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

リビングヒト臍帯静脈内皮細胞における高解像度タイムラプスイメージングと微小管ダイナミクスの自動解析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

出芽酵母における高解像度画像と個人アストラル微小管動態の解析

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...