LVは、NSF（IOS 1002837）によってサポートされています

1。プロトプラストの作成<ol><li>ペトリ皿にマンニトール8％9 mLを加え。 </li><li>スパチュラを用いて、5の2-3 PPNH4プレートから7日間の古い苔を置く - ペトリ皿を含むマンニトールで。 </li><li>シャーレに2％Driselase 3mLの（シグマD9515 - 25G）を追加します。 </li><li> 1時間静かに振とうしながら室温でペトリ皿をインキュベートする。 </li><li> 100μmのメッシュ（BDファルコン352350）を通じてプロトプラスト懸濁液をフィルターします。 </li><li> 5分の250 gでフィルタリングされたサスペンションを回転させる。上清を取り除く。 </li><li> 8％マンニトール500μLのと非常に穏やかにプロトプラストを再サスペンド。 </li><li>培養チューブの9.5 mLの8％マンニトールを追加。プロトプラストが完全に中断されていることを確認します。 </li><li>ろ過と再懸濁のステップ（1.6、1.7および1.8）をさらに2回繰り返します。 </li><li>プロトプラスト溶液10μLを取り、血球計算盤でプロトプラストをカウント。 </li><li> mL当たりプロトプラストの数を取得するために、000で16㎡地域におけるプロトプラストの数を掛ける（図1を参照）。 </li></ol> 2。変換<ol><li> 5分の250 gでプロトプラスト懸濁液をスピン。上清を取り除く。 </li><li> 160万プロトプラスト/ mLの濃度でMMGの溶液中でプロトプラストを再サスペンド。 </li><li> 20分間室温でプロトプラスト懸濁液をインキュベートする。 </li><li> 60μgのDNAを含有する培養管にプロトプラスト懸濁液600μLを加える。チューブ優しくスワール。 </li><li>プロトプラスト/ DNA混合物中にPEG / Caのソリューションを700μLを加える。全体の混合物が均質になるまでゆっくりと渦巻きがチューブ。 </li><li>室温で30分間混合物をインキュベートする。 </li><li>少なくとも5分間プレートの表面に水和物へのセロファンを可能にする、この待機期間中は、PRM - Bセロファン付きプレート（80 mm径の円、AAパッケージリミテッド、英国）をカバー。 </li><li>スパチュラで、セロファンとプレートの間に閉じ込められた気泡を取り除く。 </li><li> W5溶液3 mLの混合物を希釈する。 </li><li> 5分の250 gで混合物を回転させる。上清を取り除く。 </li><li> PRM - Tの溶融を2mLで再懸プロトプラスト（この溶液は42℃で液体に保つことができるプロトプラストに追加する前に）。セロハンでカバーPRM - Bプレート当たりの再サスペンドプロトプラストのプレート1 mLの。細孔（3M、ヘルスケア）でプレートを包みます。成長チャンバー内で25℃でプレートを保管してください。 </li><li>また、プロトプラストを液体メッキの培地とセロハンで覆われたPRM - Bプレート上にプレーティング0.5 mLを1 mLに再懸濁させることができる。これは速く再生することができますが、再生工場の数は低くなります。 </li><li>成長チャンバーは16時間に設定されています。光と8時間。暗サイクル。 </li><li> 4日間の変換後の新鮮な選択プレートに上にセロハンを移動します。 </li></ol> 3。代表的な結果： 変換の例を図2に示されています。この例で使用されているDNAは、ハイグロマイシン耐性カセットを有するプラスミドスーパーコイル7.8キロバイトでした。播種プロトプラストの量は、写真撮影のための50％に減少した。写真は、植物を選択プレートに移動された二週間後に採取された。形質転換効率は最大60μgまでのDNA濃度の影響を受けません。 DNAの高濃度では形質転換の効率に有害である。データは、このメソッドは、DNA量の広い範囲にわたって一貫性のある結果をもたらすことを示した。また、このメソッドは効果的に表1に示すように安定した形質転換体を生成することも可能です。こちらは、以前に14に報告されたものよりはるかに高い直鎖状DNAのマイクログラムあたり4安定した形質転換体を、約得ることができます。 我々はまた、マーカーとしての蛍光タンパク質の遺伝子を運ぶスーパーコイルプラスミドを形質転換して形質転換効率で熱ショックの効果を試験した。熱ショックは3分間45℃の水浴で混合物をインキュベートすることにより、10分室温でのインキュベーション（ステップ2.6以内）後に実施した。混合物を17分間熱ショック直後に室温の水浴中でインキュベートした。結果は、22時間変換後に記録した。で得られた形質転換体と熱ショックなしの比率は（〜25％）非常に類似していたが、我々は、プロトプラスト（データは示さず）の生存に熱ショックの悪影響を観察した。 <img src="/files/ftp_upload/2560/2560fig1.jpg" alt="図1" /> 図1。血球計算板のプロトプラストの表示。プロトプラストの計数のために必要なセクションが赤い四角で示されます。プロトプラストをカウントするために必要な16の正方形の点に注意してください（1.11参照）、画像は24プロトプラスト（240,000細胞/ mL）の数を示しています。 <img src="/files/ftp_upload/2560/2560fig2.jpg" alt="図2" /> 図2。 18日間の古い一時的な形質転換体の写真。プロトプラストのトランスいたスーパーコイル状プラスミドの指示された量で形成。 ：15μg、B：30μg、C：60μg、D：120μg。 <table border="1"><tbody><tr><td> DNAの量（μg） </td><td> 効率（tranformants /μgのDNA） </td></tr><tr><td> 15は、プラスミドスーパーコイル</td><td> 120 ± 24 </td></tr><tr><td> 30は、プラスミドスーパーコイル</td><td> 145 ± 54 </td></tr><tr><td> 60は、プラスミドスーパーコイル</td><td> 121 ± 60 </td></tr><tr><td> 120は、プラスミドスーパーコイル</td><td> 71 ± 38 </td></tr><tr><td> 60は、線状化プラスミド</td><td> 4 ± 1 </td></tr></tbody></table>種々のDNA量で表1。形質転換効率。

<ol>
	<li>Abel, S., Theologis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+transformation+of+Arabidopsis+leaf+protoplasts:+a+versatile+experimental+system+to+study+gene+expression.">Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression.</a> Plant J. 5, 421-427 (1994).</li><li>Wood, A. J., Oliver, M. J., Cove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bryophytes+as+model+systems.">Bryophytes as model systems.</a> Bryologist. 103, 128-133 (2000).</li><li>Schaefer, D. G., Zr&#255;d, J. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+targeting+in+the+moss+Physcomitrella+patens.">Efficient gene targeting in the moss Physcomitrella patens.</a> Plant J. 11, 1195-1206 (1997).</li><li>Schaefer, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+targeting+in+Physcomitrella+patens.">Gene targeting in Physcomitrella patens.</a> Curr. Opin. Plant Biol. 4, 143-150 (2001).</li><li>Kamisugi, Y., Cuming, A. C., Cove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parameters+determining+the+efficiency+of+gene+targeting+in+the+moss+Physcomitrella+patens.">Parameters determining the efficiency of gene targeting in the moss Physcomitrella patens.</a> Nucleic Acids Res. 33, e173-e173 (2005).</li><li>Cove, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+moss+Physcomitrella+patens.">The moss Physcomitrella patens.</a> Annu. Rev. Genet. 39, 339-358 (2005).</li><li>Kamisugi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+gene+targeting+in+Physcomitrella+patens:+homologous+recombination,+concatenation+and+multiple+integration.">The mechanism of gene targeting in Physcomitrella patens: homologous recombination, concatenation and multiple integration.</a> Nucleic Acids Res. 34, 6205-6215 (2006).</li><li>Vidali, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+formin-mediated+actin-filament+elongation+is+essential+for+polarized+plant+cell+growth.">Rapid formin-mediated actin-filament elongation is essential for polarized plant cell growth.</a> Proc. Natl. Acad. Sci. U.S.A. , (2009).</li><li>Sawahel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+foreign+DNA+into+Physcomitrella+protonemal+tissue+by+using+the+gene+gun.">Transfer of foreign DNA into Physcomitrella protonemal tissue by using the gene gun.</a> Plant Mol. Biol. 10, 315-316 (1992).</li><li>Schaefer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transformation+of+the+moss+Physcomitrella+patens.">Stable transformation of the moss Physcomitrella patens.</a> Mol. Gen. Genet. 226, 418-424 (1991).</li><li>Cho, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+bombardment+mediated+transformation+and+GFP+expression+in+the+moss+Physcomitrella+patens.">Particle bombardment mediated transformation and GFP expression in the moss Physcomitrella patens.</a> Mol. Cells. 9, 14-19 (1999).</li><li>Ashton, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bryophyte+Physcomitrella+patens+replicates+extrachromosomal+transgenic+elements.">The bryophyte Physcomitrella patens replicates extrachromosomal transgenic elements.</a> New Phytol. 146, 391-402 (2000).</li><li>Hohe, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+and+highly+standardized+transformation+procedure+allows+efficient+production+of+single+and+multiple+targeted+gene-knockouts+in+a+moss,+Physcomitrella+patens.">An improved and highly standardized transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens.</a> Curr. Genet. 44, 339-347 (2004).</li><li>Vidali, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profilin+is+essential+for+tip+growth+in+the+moss+Physcomitrella+patens.">Profilin is essential for tip growth in the moss Physcomitrella patens.</a> Plant Cell. 19, 3705-3722 (2007).</li></ol>

利害の衝突は宣言されません。

ここで紹介する方法は、 ヒメツリガネゴケのプロトプラストへの単純で一貫性DNA形質転換が可能になります。このメソッドはもともとシロイヌナズナ 1の開発が、ここではそれがヒメツリガネゴケ 、簡単で異なる植物のプロトプラストとよく実行することが実証された。したがって、この方法は若干変更して他の植物種に適用することができます。最適化のための実行可能な変更は、MMGとPEG /カルシウム溶液中でのMMGのプロトプラストの濃度、インキュベーション時間が含まれています。形質転換体の数がこの時点（表1）までのDNA濃度に比例して増加するため、我々は、ルーチンの実験で60μgのDNAを使用することを選択。我々は、植物の大規模な人口のさらなるスクリーニングと分析ができるように、単一の60μgのDNA形質転換、上の約7000の過渡形質転換体または200安定な形質転換体を得ることができる。 変換されたプロトプラストを液体メッキ媒体またはPRM - Tで拡散することができるのどちらか。再生効率が液体メッキの培地が使用されているときに低いですがプロトプラストをスーパーコイル状のプラスミドで形質転換されたとき、我々はこの方法を選択してください。表1に示した、スーパーコイルプラスミドDNAで行う変換は、より多くの形質転換体を生成し、したがって、我々はまだ液体メッキの培地で植物の多数を得ることができます。さらに、液体メッキ培地でプロトプラストを分散するとの実験9,15をノックダウンするようなRNAiのような一時的な表現型を、関連する実験のために重要である高速な再生を可能にする。さらに、トップアガーがない場合は、免疫染色とRT - PCR 9,15のための簡単な植物を分離することができます。

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 成分 </td></tr><tr><td> PPNH 4 </td><td> 1.84 mMのKH 2 PO 4 3.4 mMのカルシウム（NO 3）2 1 mMのMgSO 4を 2.72 mMの二アンモニウム、酒石酸 54μMのFeSO 4 · 7H 2 O 9.93μMH 3 BO 3 1.97μMMnCl 2を 0.4 H 2 O 0.23μMのCoCl 2 · 6H 2 O 0.19μMZnSO 4。 7H 2 O 0.22μmののCuSO 4。 5H 2 O 0.10μMのNa 2酸化モリブデン4 · 2H 2 O 0.168μMのKI 固体培地は0.7％の寒天を追加。 </td></tr><tr><td> PRM - B </td><td> 6％マンニトール、0.8％の寒天とPPNH 4。プレートを注ぐ前に、10mLの1MのCaCl 2から1 Lを追加。 </td></tr><tr><td> PRM - T </td><td> 6％のマンニトール及び0.6％寒天とPPNH 4。使用前に0.5mLの1MのCaCl 2に50mLを追加。 </td></tr><tr><td>液体めっきミディアム</td><td>寒天なしで8.5％マンニトールとPPNH 4。使用前に0.5mLの1MのCaCl 2に50mLを追加。 </td></tr><tr><td> 2％Driselase </td><td> 8％マンニトール200mlにDriselase 4gの（シグマD9515 - 25G）を追加します。 30分間静かにかき混ぜる。室温で30分間インキュベートする。 4℃、5分間穏やかに撹拌する。室温で。 10分間2500グラムをスピン、ペレットを捨てる。 10mLのアリコートとして凍結さ0.22μmの、そして店舗でフィルタ。使用前に室温の水浴で融解。 </td></tr><tr><td> MMG </td><td> 0.4 Mマンニトール 15mMのMgCl 2の 4mMのMES（pHは5.7） </td></tr><tr><td> PEG / Ca比</td><td> 4グラムPEG4000 3 mLのH 2 O 2.5mLの0.8 Mマンニトール 1 mLの1M CaCl 2を </td></tr><tr><td> W5 </td><td> 154 mM NaClを 125ミリメートルのCaCl 2 5mMのKClを 2 mMのMES（pHは5.7） </td></tr></tbody></table>

efficient polyethylene glycol (peg) mediated transformation of the moss physcomitrella patens

<p class="jove_content"&gt;変換するのは簡単かつ効率的な方法は、<em&gt;ヒメツリガネゴケpantens</em&gt;プロトプラストが記載されている。このメソッドは、のためのプロトコルから適応される<em&gt; Physocmitrella</em&gt; protonemalプロトプラストと<em&gt;シロイヌナズナ</em&gt;葉肉プロトプラスト形質転換<sup&gt; 1</sup&gt;。相同組換え効率的分裂を受けるためにその能力に起因する<em&gt;、ヒメツリガネゴケ</em&gt;は、近年、重要なモデル系として注目されています<sup&gt; 2</sup&gt;。この能力は、遺伝子の高い周波数がターゲット可能に<sup&gt; 3月9日</supなどの他のモデル植物で見ていない&gt;、<em&gt;シロイヌナズナ</em&gt;。このシステムを最大限に活用するには、我々は、コケの細胞にDNAを提供する効果的かつ簡単な方法が必要。このコケを変換する最も一般的な方法は、粒子衝突です。<sup&gt; 10</sup&gt;とPEG -媒介DNA取り込み<sup&gt; 11</sup&gt;。粒子衝突は、高い形質転換効率を生成することができますが、<sup&gt; 12</sup&gt;、遺伝子銃は多くの研究室へ容易に入手できないとプロトコルが標準化することは困難です。一方、PEG媒介形質転換は、特殊な機器を必要としない、そして無菌フードを持つ任意の実験室で行うことができます。ここでは、コケのプロトプラストの形質転換のための簡単​​かつ非常に効率的な方法を示しています。このメソッドは、以前に報告された最も効率的なプロトコルから改善されているDNAのマイクログラムあたり120以上の一過性形質転換体を、生成することができます<sup&gt; 13</sup&gt;。そのシンプルさ、効率性、および再現性のため、この方法は、ルーチンの変換のためだけでなく、形質転換体の大きい数を必要とするプロジェクトに適用することができます。</p

Watch this Scientific Journal Video about Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens at JoVE.com

Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens

変換するのは簡単かつ効率的な方法<em&gt;ヒメツリガネゴケpantens</em&gt;プロトプラストが記載されている。このメソッドは、のためのプロトコルから適応される<em&gt; Physocmitrella</em&gt; protonemalプロトプラストと<em&gt;シロイヌナズナ</em&gt;葉肉プロトプラスト形質転換<sup&gt; 1</sup&gt;。

効率的なポリエチレングリコール（PEG）モスの媒介形質転換ヒメツリガネゴケ</em

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella ...

efficient-polyethylene-glycol-peg-mediated-transformation-moss

Research

JoVE Journal

Biology

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

40.5K ビュー.  Worcester Polytechnic Institute- WPI. 変換するのは簡単かつ効率的な方法ヒメツリガネゴケpantensプロトプラストが記載されている。このメソッドは、のためのプロトコルから適応される Physocmitrella protonemalプロトプラストとシロイヌナズナ葉肉プロトプラスト形質転換 1。

ビデオ: Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens

Environmentally Induced Heritable Changes in Flax

Some flax varieties respond to nutrient stress by modifying their genome and these modifications can be inherited through many generations. Also associated with these genomic changes are heritable phenotypic variations 1,2. The flax variety Stormont Cirrus (Pl) when grown under three different nutrient conditions can either remain inducible (under the control conditions), or become stably modified to either the large or small genotroph by growth under high or low nutrient conditions respectively. The lines resulting from the initial growth under each of these conditions appear to grow better when grown under the same conditions in subsequent generations, notably the Pl line grows best under the control treatment indicating that the plants growing under both the high and low nutrients are under stress. One of the genomic changes that are associated with the induction of heritable changes is the appearance of an insertion element (LIS-1) 3, 4 while the plants are growing under the nutrient stress. With respect to this insertion event, the flax variety Stormont Cirrus (Pl) when grown under three different nutrient conditions can either remain unchanged (under the control conditions), have the insertion appear in all the plants (under low nutrients) and have this transmitted to the next generation, or have the insertion (or parts of it) appear but not be transmitted through generations (under high nutrients) 4. The frequency of the appearance of this insertion indicates that it is under positive selection, which is also consistent with the growth response in subsequent generations. Leaves or meristems harvested at various stages of growth are used for DNA and RNA isolation. The RNA is used to identify variation in expression associated with the various growth environments and/or t he presence/absence of LIS-1. The isolated DNA is used to identify those plants in which the insertion has occurred.

Growing some flax varieties under nutrient stress results in genomic variation within a subset of the genome and phenotypic variation. A complex insertion at a specific site is associated with growth under various nutrient regimes and with changes in gene expression around this site.

環境的に誘導されたフラックスの変化を遺伝

Some flax varieties respond to nutrient stress by modifying their genome and these modifications can be inherited through many generations. Also ...

生物学

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s1, then followed by vitrification methods developed in the late 1980s2. Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4&deg;C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained3, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature4.
Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos5. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.

An ethylene glycol-based vitrification method for mouse embryos is described. It is advantageous to other methods in its simplicity and low embryonic toxicity, and therefore can be broadly applicable to many strains of mice, including inbred and gene-modified mice.

エチレングリコール系ガラス化によるマウス胚の凍結保存

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

Picoeukaryoteのゲノム形質転換<em&gt; Ostreococcusタウリ</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

組換えタンパク質の高レベルの一過性発現のための植物の効率的なアグロ

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

エピソームおよびHDAC阻害剤を用いて血液からの効率的なiPS細胞の生成

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines.

アグロバクテリウム-形態新生遺伝子を用いた再カルシトランストウモロコシ近交線の媒介性不成熟胚変態

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

CRISPR/Cas9を使用してヌル突然変異を生成するための Culex 蚊の胚の調製と注入

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2&#8211;3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

This work describes the use of CRISPR-Cas9 genome editing technology to knockout endogenous gene OsABCG15 followed by a modified Agrobacterium-mediated transformation protocol to produce a stable male-sterile line in rice.

アグロバクテリウム-媒介遺伝子変換、トランスジェニック産生、および米における男性生殖発達の研究への応用

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive ...

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic pest management strategies. The most used transposable element in insect transformation is piggyBac, and piggyBac-based germline transformation has been successfully conducted in model insects. However, it is still challenging to employ this technology in non-model insects that include agricultural pests. This paper reports on germline transformation of a global agricultural pest, the fall armyworm (FAW), Spodoptera frugiperda, using the hyperactive piggyBac transposase (hyPBase).
In this work, the hyPBase mRNA was produced and used in place of helper plasmid in embryo microinjections. This change led to the successful generation of transgenic FAW. Furthermore, the methods of screening transgenic animals, PCR-based rapid detection of transgene insertion, and thermal asymmetric interlaced PCR (TAIL-PCR)-based determination of the integration site, are also described. Thus, this paper presents a protocol to produce transgenic FAW, which will facilitate piggyBac-based transgenesis in FAW and other lepidopteran insects.

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda

Successful germline transformation in the fall armyworm, Spodoptera frugiperda, was achieved using mRNA of hyperactive piggyBac transposase.

秋ミミミズ、スポドプテラ・フルギペルダにおける超活性ピギーバツトランスポザーゼ媒介性生殖細胞変容

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic ...

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is a newly isolated filamentous cyanobacterium. This paper describes how new filamentous cyanobacteria can be isolated from marine rockpools. It also describes how DNA can be extracted from filaments and how the genomes can be sequenced. Although transformation is established for many single-celled species, it is less frequently reported for filamentous cyanobacteria. A simplified method for the natural transformation of P. lacuna is described here. P. lacuna is the only member of the order Oscillatoriales for which natural transformation is established. This paper also shows how natural transformation is used to express superfolder green fluorescent protein (sfGFP). An endogenous cpcB promoter induced approximately 5 times stronger expression than cpc560, A2813, or psbA2 promoters from Synechocystis sp. PCC6803. Further, a method for the cryopreservation of P. lacuna and Synechocystis sp. CPP 6803 was established, and methods for assessing motility in a liquid medium and on agar and plastic surfaces are described.

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Phormidium lacuna is a filamentous cyanobacterium that was isolated from marine rockpools. This article describes the isolation of filaments from natural sources, DNA extraction, genome sequencing, natural transformation, expression of sfGFP, cryoconservation, and motility methods.

糸状シアノバクテリウム・ホルミジウム・ラクナの自然形質転換、タンパク質発現、凍結保存

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is ...