この作品は、全米科学財団 (NSF-PGRP IOS-1339388) から植物ゲノム研究プログラムによって支えられました。

1. 植物の成長
<ol><li>大豆の 5-10 ダイズ種子 (ウィリアムズ 82) 長日条件 (16 h 1,500 µmol m-2の-1光) カスタム土壌ミックスの 25 ° c の下で温室で 13 cm ポットに種をまく (、1: 土、パーライト、魚雷の砂の比率 1:1)。</li>
</ol>2. DNA のプラスミッドの準備
<ol><li>滅菌ピペット チップやつまようじを使用すると、単一コロニーを大腸菌興味の遺伝子を含んでいるプラスミッドを運ぶ冷凍グリセロール ストックを選ぶし、20 mL ルリア ベルターニカミラ (LB) 液体培地 50 mL のフラスコに適切な抗生物質で予防接種します。</li>
	<li>シェーカーで一晩 37 ° C でフラスコを孵化させなさい。5 分間室温で 12,000 × g の懸濁液を遠心分離し、上清を廃棄して細菌を収集します。抽出・精製プラスミド プラスミド準備キットの製造元の手順を実行します。</li>
</ol>3. プロトプ ラスト単離
<ol><li>10 日間の古い大豆苗 (図 1) から新しく拡張された単葉の葉をカットします。 注: 適切な発達段階で葉の選択は大豆プロトプ ラスト調製成功への鍵です。初期の発達段階で展開直後の葉だけ使用します。成熟した葉、消化する細胞壁と難しくなってください。</li>
	<li>新鮮なかみそりの刃を含む葉と 0.5-1 に残る mm ストリップ、カットから主脈を削除します。 注: 2 つの単葉の葉 10 mL の酵素液で消化は以上 10 変換のため十分なプロトプ ラストを与えます。</li>
	<li>葉が 10 mL を 15 mL チューブに作りたての酵素液 (表 1) にすぐにそして軽くをストリップ鉗子、転送のペアを使用しています。真空潜入葉ストリップ部屋の温度で 15 分。</li>
	<li>室温で 4-6 h の低照度下で穏やかな攪拌 (40 回転) と酵素液で葉の細片を孵化させなさい。プロトプ ラストがリリースされると、酵素液が黄色緑を回すことを確認します。(X10) 顕微鏡下で酵素/プロトプ ラスト ソリューションを確認してください。 注: リリースのプロトプ ラストは球状形、未消化の細胞が不規則なまたは楕円形です。</li>
	<li>そっとして 50 mL のチューブで酵素/プロトプ ラスト溶液 10 mL を転送し、消化を停止するために室温 W5 溶液 (表 1) 10 mL を追加します。穏やかに数回チューブを反転します。未消化の葉身を削除する 50 mL のチューブの上に置かれたきれいな 75 μ m ナイロン メッシュに酵素/プロトプ ラストの溶液を注ぐ穏やか。</li>
	<li>室温で 1-2 分の 50 mL のチューブで 100 x g でプロトプ ラスト酵素/フロースルー ソリューションを遠心します。プロトプ ラストのペレットを乱すことがなく 10 mL の血清ピペットを使用して上清を慎重に取り外します。</li>
	<li>プロトプ ラストを再懸濁し、プロトプ ラスト (x10) 顕微鏡で血球の数を数えることによって 2 x 105 mL-1 4 ° C に冷やした W5 ソリューションでの濃度に希釈します。30 分間氷の上プロトプ ラストをしてください。</li>
	<li>室温で 1-2 分の 100 x グラムの懸濁液を遠心し、原形質体の餌を乱すことがなく 1 mL ピペットを使用した W5 ソリューションを慎重に取り外します。室温で 2 x 105 mL-1濃度で MMG 解決方法 (表 1) プロトプ ラストを再懸濁します。</li>
</ol>4. プロトプ ラスト
<ol><li>プロトプ ラスト (2 x 104プロトプ ラスト 2 x 105 mL-1) の 100 μ 因数は 1.5 mL 低接着マイクロ遠心チューブ用ノーカット 200 μ L ピペット チップを使用してします。ネガティブ コントロールとして機能する 1 つの因数脇を置きます。プロトプ ラストの残りの因数のそれぞれにプラスミド (10-20 μ g) の 10 μ L を追加します。</li>
	<li>ゆっくり 1.5 mL 遠心チューブの内壁に作りたての止め釘の解決 (表 1) の 110 μ L を追加し、軽く反転ソリューションが均質になるまでチューブを回転させてください。</li>
	<li>15 分間室温で変換の混合物を孵化させなさい。</li>
	<li>変換を停止するには、ゆっくりと室温で 1.5 mL チューブに W5 ソリューションの 400 μ L を追加し、ソリューションが均一になるまでやさしくチューブを転倒させます。室温で 1-2 分の 100 g でチューブを遠心分離、ピペットを使用して上澄みを廃棄します。</li>
	<li>チューブに無線ソリューション (表 1) の 1 つの mL を追加し、1-2 回を軽くピペッティングによって再懸濁します。表面をコートし、プロトプ ラストが板にくっつかないように 6 ウェル培養プレートの各ウェルに 5% (巻/巻) 滅菌子牛血清 1 mL を追加します。</li>
	<li>数秒後、ピペットを使用して子牛血清を破棄します。再懸濁のプロトプ ラスト培養プレートのウェルに移転します。蓋付きプレートをカバーします。</li>
</ol>5. プロトプ ラストの培養と収穫
<ol><li>暗闇の中で 1-2 日間室温でプロトプ ラストを孵化させなさい。 注: 1 日培養に比べて一般に強い蛍光蛋白質信号を 2 日間培養に利回りし、蛍光蛋白質信号が 3-4 日間続く可能性がありますがわかった。</li>
	<li>プロトプ ラストのソリューションを 1.5 mL の低接着微量遠心チューブに転送します。プロトプ ラストを収穫するために室温で 1-2 分の 100 x g でチューブを遠心します。ピペットを使用して上清を除去し、スライド ガラスに 10 μ L プロトプ ラストを転送します。</li>
	<li>蛍光共焦点顕微鏡又は蛍光信号を観察します。(信号を守らなければなりませんない) 陰性対照として非変形プロトプ ラストを使用します。</li>
</ol>

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A., 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=The+NAC+domain-containing+protein,+GmNAC6,+is+a+downstream+component+of+the+ER+stress-and+osmotic+stress-induced+NRP-mediated+cell-death+signaling+pathway.">The NAC domain-containing protein, GmNAC6, is a downstream component of the ER stress-and osmotic stress-induced NRP-mediated cell-death signaling pathway.</a> BMC Plant Biol. 11 (1), 129 (2011).</li><li>Kidokoro, 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=Soybean+DREB1/CBF-type+transcription+factors+function+in+heat+and+drought+as+well+as+cold+stress-responsive+gene+expression.">Soybean DREB1/CBF-type transcription factors function in heat and drought as well as cold stress-responsive gene expression.</a> Plant J. 81 (3), 505-518 (2015).</li><li>Sun, X., 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=Targeted+mutagenesis+in+soybean+using+the+CRISPR-Cas9+system.">Targeted mutagenesis in soybean using the CRISPR-Cas9 system.</a> Sci Rep-UK. 5, 10342 (2015).</li><li>Karimi, M., Inz&#233;, D., Depicker, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GATEWAY&#8482;+vectors+for+Agrobacterium-mediated+plant+transformation.">GATEWAY&#8482; vectors for Agrobacterium-mediated plant transformation.</a> Trends Plant Sci. 7 (5), 193-195 (2002).</li><li>Xia, Z., 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=Positional+cloning+and+characterization+reveal+the+molecular+basis+for+soybean+maturity+locus+E1+that+regulates+photoperiodic+flowering.">Positional cloning and characterization reveal the molecular basis for soybean maturity locus E1 that regulates photoperiodic flowering.</a> P. Natl. A. Sci. USA. 109 (32), E2155-E2164 (2012).</li></ol>

著者は、彼らは競合する金銭的な利益があることを宣言します。

ダイズプロトプと一過性発現研究への応用の分離のこのプロトコルは徹底的にテストされており、当研究室では非常にうまきます。手順はシンプルで簡単な一般的な機器を最小コストを必要とされます。我々 のプロトコルは、以前に報告された方法34,35,36,37,38に比べて均一で高品質のプロトプ ラストの大量を生成します。ただし、プロトプ ラストの収率と変換レートに影響を与える多くの要因があるので、実験条件、望ましい結果および使用される材料に従って条件を最適化する研究者も強く推奨します。このシステムは植物細胞における即時規制および生化学的イベントの検討に非常に役立ちますが、組織または個体レベルで発生するイベントと長期的細胞プロセスの観察に適してはありません。
理想的な発達段階で成長する植物材料の選択だった大豆プロトプ ラスト調製で最も重要な要因であることがわかった。プロトプ ラスト、しかしその後 DNA 変換に影響を与える品質の利回りだけでなくを決定します。常にストレスを感じないで、干ばつ、洪水、極端な温度や害虫などから一定の環境で大豆の植物を育てることが重要です。最高のプロトプ ラスト収量と変換効率は、稚苗からちょうど拡大された単葉の葉を使用して実現できます。初心者は、発達段階別に単葉の葉を使用し、最高の結果を得るために比較することをお勧めします。
プロトプ ラスト/DNA 比は、最適な変換効率を上げるのための重要な要因です。手始めに 2 × 104プロトプ ラスト/10-20 μ g の DNA 比、個々 の構成要素の比率の最適化お勧め25通常お勧めします。高品質 DNA 精製キットによって得られた DNA を使用することを強くお勧めします。
プロトプ ラストにおける過渡的表現のためのベクトルの選択、高コピー数と小型ベクトルが一般に好ましいです。バイナリのアグロバクテリウムのベクトルが-より少ない最適な変換効率これらのベクトルの大きいサイズがあります、プロトプ ラストの植物を介した変換を使用できます。GFP 融合タンパク質発現、我々 は通常 p2GWF7 と p2FGW739 (https://gateway.psb.ugent.be) など植物あり、従って比較的小さい (6-7 kb)、選択マーカーを持っていないベクトルを使用します。
複数のベクトルは、プロトプ ラストを同時に変換する使用できます。オルガネラとダイズプロトプ ラストの細胞内ラベリングのための複数の蛍光蛋白質と同様、蛋白質蛋白質の相互作用をテストするため BiFC ベクトルを表現することに成功しました。シンプルさと手法の大規模な利回りが規制や生化学的イベント、標的遺伝子ベクター、一過性に表現された蛋白質と蛋白質の複合体の分離の検討そして特定の浄化のための理想的なシステムを提供していますさらに、新興 genomic および proteomic アプリケーションがさらにできるように核などの細胞小器官に近づきます。

プロトプ ラストは、削除される細胞壁を持つ植物細胞です。機能のほとんどは、植物細胞の活動を管理するのでプロトプ ラスト観察・多様な携帯電話イベントを評価する良いモデル系、1体の交配を研究し、植物の再生2する貴重なツールです。プロトプ ラストは、細胞壁がブロックされセルに DNA の通過以来工場変換3,4、5、また広く利用されています。プロトプ ラストは生理学的な応答のいくつか、したがって細胞内タンパク質局在6,7、8を研究する基礎研究の基本的な値を提供して、そのまま植物の細胞プロセスを所有しています。蛋白質蛋白質の相互作用の9,10、プロモーター活動11,12,13ではセルを住んでいます。
196014で最初に報告された植物プロトプ ラストの分離と分離とプロトプ ラストの変形のためのプロトコルを開発し、最適化します。葉の切断と非消化組織破片からリリースされた原形質体の分離に続いて、細胞壁の酵素消化、原形質体の隔離の標準的な手順が含まれます。エレクトロポレーション15,16、マイクロインジェクション17,18、およびポリエチレング リコール (PEG)4,5,19手法変革戦略が含まれています。種の広い範囲は、原形質体の隔離、柑橘類20、アブラナ科21、ナス科22その他の観賞用植物家族23,24を含む成功報告されています。モデル植物シロイヌナズナの葉から分離したシロイヌナズナ葉肉プロトプ ラスト (TEAMP) に一過性発現系が確立した25をされている様々 な種では、多様な組織型が使用されて、多様なアプリケーションに広く採用されています。
大豆 (最高グリシン(l.)ダイズ) 最も重要なタンパク質の 1 つで、油作物26。異なり、シロイヌナズナイネ、遺伝子組み換え大豆の植物を得ることはむしろ困難と低効率に知られています。アグロバクテリウム-仲介された浸潤一般に使用されているタバコ27で表皮細胞およびシロイヌナズナ28,29, 苗の一時的な遺伝子発現研究に対しサツマイモネコブセンチュウは大豆30植物毛状根の変形のために使用されています。ウイルス誘導性遺伝子のサイレンシング アプローチは、ターゲット遺伝子の31,32と過渡式の33のダウンレギュレーションを系統的に利用されています。プロトプ ラストは、貴重な汎用性の高いこれらのアプローチ方法を提供します。プロトプ ラストは大豆の地上材料から得ることが、発現の迅速かつ同期ができます。しかし、1983年34ダイズプロトプ ラストの最初の成功した分離以来報告されている限られた大豆35,36,37,プロトプ ラストの応用38、主にダイズプロトプ ラストの比較的低い収穫のため。
ここでは、ダイズプロトプ ラストの分離のためのシンプルで効率的なプロトコルと一時的な遺伝子発現研究への応用について述べる。ダイズ芽ばえから若い単葉の葉を使用すると、数時間以内の重要なプロトプ ラストの大量を取得することができました。さらに、ペグによるカルシウムの変換法はシンプルで高効率で大豆プロトプ ラストに DNA を提供する低コストを最適化しています。

<table border="0" cellpadding="0" cellspacing="0" width="723">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">MES</td>
			<td style="width:181px;">Sigma Aldrich&#160;</td>
			<td style="width:119px;">M8250-100G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cellulase CELF</td>
			<td>Worthington Biological Corporation</td>
			<td>LS002611</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pectolyase Y-23</td>
			<td>BioWorld</td>
			<td>9033-35-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CELLULASE &#34;ONOZUKA&#34; R-10</td>
			<td>yakult</td>
			<td>10g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACEROZYME R-10</td>
			<td>yakult</td>
			<td>10g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mannitol</td>
			<td>ICN Biomedicals&#160;</td>
			<td>152540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Fisher&#160;</td>
			<td>C79-500g&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td>NEB</td>
			<td>R3535S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DTT</td>
			<td>Sigma Aldrich&#160;</td>
			<td>D5545-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Sigma Aldrich&#160;</td>
			<td>S7653-1kg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>Fisher&#160;</td>
			<td>P217-500g&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2</td>
			<td>Sigma Aldrich&#160;</td>
			<td>M8266-100g</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PEG4000</td>
			<td>Fluka</td>
			<td>81240</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nylon mesh</td>
			<td>carolina</td>
			<td>652222N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Tissue Culture Plates&#160;</td>
			<td>USA Scientific</td>
			<td>CC7682-7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Razor Blades</td>
			<td style="width:181px;">Fisher</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hemacytometer</td>
			<td>hausserscientific</td>
			<td>1483</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">QIAprep Spin Miniprep Kit</td>
			<td style="width:181px;">Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">EZNA plasmid miniprep kit</td>
			<td style="width:181px;">Omega</td>
			<td>D6942-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">GeneJET Plasmid Miniprep Kit</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td>K0502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Centrifuge 5810</td>
			<td style="width:181px;">eppendorf</td>
			<td style="width:119px;">5811000827</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Centrifuge 5424</td>
			<td style="width:181px;">eppendorf</td>
			<td>22620401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Jencons Powerpette Plus Pipet Controller</td>
			<td style="width:181px;">Jencons</td>
			<td>14526-202</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:25px;width:256px;">Zeiss 710 Confocal Microscope</td>
			<td style="width:181px;">Zeiss</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:256px;">Nonstick, RNase-free Microfuge Tubes, 1.5 mL</td>
			<td style="width:181px;">Ambion</td>
			<td style="width:119px;">AM12450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">15 mL Centrifuge Tubes</td>
			<td style="width:181px;">Denville</td>
			<td style="width:119px;">C1018-P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">50 mL Centrifuge Tubes</td>
			<td style="width:181px;">Denville</td>
			<td style="width:119px;">C1060-P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Newborn Calf Serum</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td style="width:119px;">16010159</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Soil</td>
			<td style="width:181px;">Ingram&#39;s Nursery</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">perlite</td>
			<td style="width:181px;">Vigoro</td>
			<td style="width:119px;">100521091</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">Torpedo Sand</td>
			<td style="width:181px;">JKS Ventures</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:256px;">LB Broth, Lennox (Powder)</td>
			<td style="width:181px;">Fisher</td>
			<td style="width:119px;">BP1427-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

a simple method for isolation of soybean protoplasts and application to transient gene expression analyses

大豆 (最高グリシン(l.)ダイズ) は重要な作物を遺伝学的、生化学的経路の研究マメ科のモデルとなっています。そのため、大豆の効率的な一過的遺伝子発現系を確立することが重要です。ダイズプロトプ ラストの準備のための単純なプロトコルと非定常の機能解析への応用を報告します。ダイズ芽ばえから若い単葉の葉が高品質のプロトプ ラストの大量に起因したと分かった。ペグ カルシウム仲介された変形方法を最適化することにより、大豆含むプロトプ ラストを用いた高い変換効率を達成しました。このシステムは、ライブ ダイズ細胞における複雑な規制とシグナル伝達メカニズムの検討の効率的かつ汎用性の高いモデルを提供し、ヘルプは、マメ科植物の多様な細胞・発達・生理学的なプロセスを理解するかもしれない。

プロトプ ラストの調製 (図 1) を 10 日間の古い大豆のさまざまな器官を調べた (図 2) 顕微鏡下では認められなかった.胚軸, 上胚軸から細胞壁がほとんど消化して、そしていくつかの細胞 (図 2 b, 2 C) 互いに接続されています。子葉 (図 2 D) とルート (図 2 a) は、細胞壁は細胞のごく一部でのみ削除されました。対照的に、多数プロトプ ラストの含むの頃にみ (図 2 eG) を使用します。発達段階別に単葉の葉はさらに調べた (図 2 H).展開されていないとだけ拡張含む葉プロトプ ラストの高収率の結果が生じ, 含む展開直後からのプロトプ ラストのサイズが多かった制服 (図 2 f) の未展開含む (図 2 e) より。完全に展開された含む、細胞壁は、まだそのままの細胞 (図 2)。我々 は 3 つの異なった製造業者からの細胞壁分解酵素をテストし、上記のように同等の結果を得られます。これらの観察に基づいて、植物材料の選択が重要な要因であるとプロトプ ラストの準備のための最もよい材料をエダマメ苗からちょうど拡大された単葉の葉にいたと考えられました。
プラスミド DNA (0.1 μ、1 μ、5 μ g、20 μ g) 量の範囲は、大豆含むプロトプ ラスト (図 3 a~ D) の最適な変換効率のテストされました。20 μ g のプラスミド DNA 0.1 μ g を示した最低 50% 変換レート (図 3 D) より多くの最高の変換効率を示した (1% 未満)(図 3 a)。3 つのメーカーから別の DNA 精製キットを使用して同等の変換効率を得られます。プラスミド DNA の量が多くて大きく変換効率を向上させる助けることが示唆されました。
図 4は、構成 p2GWF7-E1、 GFPマメ科特異遺伝子E1 (Glyma.06G207800)、ベクトル p2GWF7 CaMV 35Sプロモータ主導に融合を表現すると変換ダイズプロトプ ラストの共焦点画像39. E1 GFP 融合タンパク質はシロイヌナズナ プロトプ ラスト システム40を使用して前の調査に一貫している、ダイズプロトプ ラストの核局在化を示します。E1は、マメ科植物に固有の遺伝子は、大豆のプロトプ ラストを用いた結果は E1 蛋白質の細胞レベル下のローカリゼーションの決定的な洞察力を提供します。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57258/57258fig1.jpg"> 図 1。この研究では、ルート、胚軸、子葉、芽生えを含む原形質体の準備のためテストがダイズ幼植物の器官を示す図含むと。単葉の葉のペア後、は、大豆の苗は三葉の葉を開発します。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57258/57258fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57258/57258fig2.jpg"> 図 2。さまざまな臓器と大豆苗の段階から調製したプロトプ ラストの細胞します。(A ~ D)ダイズ芽ばえの 10 日間の古いさまざまな器官から調製した細胞: ルート (A)、胚軸 (B)、(C)、芽生え子葉 (D)。(E ・ G)発達段階別に単葉の葉から調製したプロトプ ラストの細胞: 未展開 (E)、(F) を発展させただけと (G) を完全に展開含む、左・中・右、上 (H) 大豆苗にそれぞれ対応します。スケール バーは 25 μ m (A G) または 25 mm (H です)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57258/57258fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57258/57258fig3.jpg"> 図 3。共焦点画像を含むプロトプ ラスト プラスミッド DNA の量が異なると大豆の変換効率を示します。画像 (緑色で表される) GFP と明るいフィールド (グレー) がマージされます。プロトプ ラストは、プラスミド p2GWF7 E1 の量が異なると変形した: 0.1 μ g (A)、(B) 1 μ、5 μ g (C)、20 μ g (D)。GFP の蛍光信号は、顕微鏡下で変換後 24 時間監視されました。ブラック スケール バーは 50 μ m います<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57258/57258fig3large.jpg" target="_blank">この図の拡大版を表示するにはここをクリックしてください。</a> 。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57258/57258fig4.jpg"> 図 4。共焦点画像含むプロトプ ラスト大豆の核に E1 GFP の細胞内分布を示すします。プラスミド p2GWF7 E1 は、変換に使用されました。画像 (緑色で表される) GFP と明るいフィールド (グレー) がマージされます。GFP の蛍光信号は変換後 24 時間監視されました。ブラック スケール バーは 5 μ m です<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57258/57258fig4large.jpg" target="_blank">この図の拡大版を表示するにはここをクリックしてください。</a> 。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td colspan="2">酵素液 (新鮮な)</td>
		</tr>
<tr>
<td>MES の pH 5.7</td>
			<td>20 mM</td>
		</tr>
<tr>
<td>セルラーゼ CELF</td>
			<td>2% (w/v)</td>
		</tr>
<tr>
<td>ペクトリアーゼ Y-23</td>
			<td>0.1% (w/v)</td>
		</tr>
<tr>
<td>マンニトール</td>
			<td>0.75 M</td>
		</tr>
<tr>
<td>CaCl2
</td>
			<td>0.2 mM</td>
		</tr>
<tr>
<td>BSA</td>
			<td>0.1% (w/v)</td>
		</tr>
<tr>
<td>DTT</td>
			<td>0.5 mM</td>
		</tr>
<tr>
<td colspan="2">W5 ソリューション</td>
		</tr>
<tr>
<td>塩化ナトリウム</td>
			<td>154 mM</td>
		</tr>
<tr>
<td>CaCl2
</td>
			<td>125 mM</td>
		</tr>
<tr>
<td>KCl</td>
			<td>5 mM</td>
		</tr>
<tr>
<td>MES の pH 5.7</td>
			<td>2 mM</td>
		</tr>
<tr>
<td colspan="2">MMg ソリューション</td>
		</tr>
<tr>
<td>MES の pH 5.7</td>
			<td>4 mM</td>
		</tr>
<tr>
<td>マンニトール</td>
			<td>400 mM</td>
		</tr>
<tr>
<td>MgCl2
</td>
			<td>15 mM</td>
		</tr>
<tr>
<td colspan="2">止め釘の解決 (新鮮な)</td>
		</tr>
<tr>
<td>PEG4000</td>
			<td>20% (w/v)</td>
		</tr>
<tr>
<td>マンニトール</td>
			<td>200 mM</td>
		</tr>
<tr>
<td>CaCl2
</td>
			<td>100 mM</td>
		</tr>
<tr>
<td colspan="2">無線ソリューション</td>
		</tr>
<tr>
<td>MES の pH 5.7</td>
			<td>4 mM</td>
		</tr>
<tr>
<td>マンニトール</td>
			<td>0.5 M</td>
		</tr>
<tr>
<td>KCl</td>
			<td>20 mM</td>
		</tr>
</tbody></table>表 1。大豆プロトプ ラスト単離と変換に使用されるソリューションです。

Watch this Scientific Journal Video about A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses at JoVE.com

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

我々 は生きているセルで複雑な規制とシグナル伝達メカニズムを研究するダイズプロトプ ラストの大量の準備のためシンプルで効率的なプロトコルを開発しました。

簡便な分離大豆プロトプ ラストと一過的遺伝子発現解析への応用

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

a-simple-method-for-isolation-soybean-protoplasts-application-to

Nanyang Technological University

Research

JoVE Journal

Biology

24.4K ビュー.  California State University, Northridge. 我々 は生きているセルで複雑な規制とシグナル伝達メカニズムを研究するダイズプロトプ ラストの大量の準備のためシンプルで効率的なプロトコルを開発しました。

ビデオ: A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

エレクトロポレーションのためのマウス骨髄からの一次造血細胞培養の準備

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

生物学

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

遺伝子発現解析のための胚ゼブラフィッシュとcDNA合成からのRNA分離

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

高効率で初代培養細胞をトランスフェクトするためにジーンパルサーMXcellのエレクトロポレーションシステムの使用

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

ハイC：ゲノムの三次元構造を研究する方法。

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

ナマルバ細胞におけるIL - 4依存性遺伝子発現のsiRNAのノックダウン：遺伝子発現研究を簡素化する自動化セルカウンターの使い方

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

のパラフィン包埋して凍結切片ショウジョウバエアダルトの筋肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

アクティブT.の試験管内再構成でcastaneumのテロメラーゼ

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

リボソーム結合した新生ポリペプチドの分離<em&gt; in vitroで</em&gt; mRNAに沿って並進一時サイトを識別するために

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

低酸素症後単一細胞レベルでのグローバルのRNA合成の分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

マウスにおける流体力学的遺伝子送達によるタンパク質の一過性発現

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

簡便な分離大豆プロトプ ラストと一過的遺伝子発現解析への応用

簡便な分離大豆プロトプラストと一過的遺伝子発現解析への応用