著者は、私たちの研究室と博士に毛状根の技術を導入するための博士クリステイラー（オハイオ州立大学）に感謝したいと思います。 Senthil Subramanianは（サウスダコタ州立大学）、プロトコルを改善するためのファン張（魯東大学、中国山東省）。我々はまた、このビデオを作るのに役立つために博士ハオチェン（南京農業大学を）感謝。この作品は、OYにDOE（DE - SC0001295）、NSF（MCB - 0923779）とUSDA（2010-65116-20514）からの助成金によって部分的にサポートされています

以下のプロトコールは、モデルマメ科植物種M.の毛状根複合植物を生成するために使用されていますtruncatula。同様のプロトコルは、少なくとも8つの植物種1〜4の適応されている。我々はMを使用truncatula毛状根複合植物は根と根粒形成における遺伝子機能を研究する。毛状根複合植物を生成する2）; 3）共生根粒菌の感染症、4）トランスジェニックのルートの識別を準備する植物材料の1）：プロトコルは、以下の4つのセクションに分けられた。我々は、複合植物のトランスジェニックルート3をスクリーニングするためのレポーターとして緑色蛍光タンパク質（GFP）遺伝子を含むバイナリーベクターを使用していました。抗生物質ベースの選択と比較すると、GFPベースのスクリーニングは、迅速、簡単、かつ安価である。私たちの構造では、ER -発現に最適化されたGFP遺伝子は、トランスジェニックおよび非トランスジェニック根の間で容易に区別ができるように、トランスジェニック根に強力な構成的GFPシグナルを持っているスーパーユビキチンプロモーターによって駆動される。 1。植物材料の準備<ol><li> M. truncautlaの種子は、温室で栽培植物（相対湿度50％、16 / 8時間の明/暗、18分の22 ° C昼/夜温）から収穫されています。成熟した種子は4℃で保存することができます</li><li>約100種が定期的に振盪しながら10分間、10mlの濃硫酸（95から98パーセント、Sigma - Aldrich社、MO）でscarifiedされています。 </li><li>種子は、穏やかに撹拌しながら滅菌水で5〜7倍をすすぐ。 </li><li>種子を6時間室温で水に浸漬し、℃で36〜48時間の発芽を同期させるために4で保持されます。 </li><li>約20〜30の種子をシャーレに入れ湿ったろ紙上に広げています。彼らは22℃に維持されています° Cを、μmol.m-2 8分の16時間明/暗サイクル、及び40で。s -1の光強度は、5-6日間。 </li><li>発芽苗が土壌に移し、月に温室内に配置されています。 </li></ol> 2。毛状根複合プラントの生成<ol><li>関心のある遺伝子を運ぶのバイナリ構造はAに変換された標準プロトコルを使用してリゾゲネス 。我々はトランスジェニック組織のスクリーニングを容易にマーカーとしてGFP遺伝子を含むバイナリーベクターのセットを設計した。これらのベクターは、種々のプロモーターを含み、すべての過剰発現または標的遺伝子の3,5のサイレンシングのためのゲートウェイのクローニング部位を（Invitrogen社、カールスバッド、カリフォルニア州）がある。 </li><li> A.リゾゲネスは 28で適切な抗生物質選択と50ミリリットルのLB培地中で16〜24時間のためのCで培養される。 </li><li>細菌は窒素を含まない植物の栄養液（表1と6）の= 0.3を採取し、OD 600の最終濃度に再懸濁されている。 </li><li>毛状根の再生をサポートするために、滅菌支持マトリックス（&quot;ロックウール&quot;、Hummertインターナショナル、アースシティ、MO）は約3 cm 3のプラグに切断される。我々一ペトリ皿に4プラグを置きます。 </li><li>穴は、外植片の挿入を容易にするためのピペットチップを使用して、各プラグの上部につついている。A.リゾゲネス （5mL）を、各プラグインに追加されます。 </li><li> 2月3日腋芽で撮影セクションは斜めカットで切り出される。外植片は、プラグに挿入し、約3週間22℃で栽培されています。我々は、5mlの窒素フリーのソリューションに必要な水、それらを、その後、最初の10日間、水遣りせずにメディの外植片を保持。我々は、頂端分裂組織を削除すると不定根の形成を減少させるかもしれないことがわかった。 </li></ol> 3。共生根粒菌（Sinorhizobium Meliloti）感染<ol><li> 3週間後、いくつかの不定根は、ロックウールから出てくる。毛状根複合プラントはロックウールの有無に関係なく、砂またはバーミキュライト（滅菌）に転送されます。彼らは22の温室で栽培されています° C、16 / 8時間の明/暗サイクル、および300μmol.m-2のもう1週間に-1光強度。 </li><li>根粒菌感染症、S.の前にmeliloti 1201は、30 ° C（表2、および6,7）で7日間に50 mlの酵母エキス-マンニトール培地中で培養する。 </li><li>再懸 ​​根粒菌の細胞は窒素フリー栄養液を用いてOD 600の最終濃度= 0.08に希釈されています。 S.の10 mlの新鮮なサスペンションmelilotiは、結節形成を誘導するために各複合プラントに適用した。 </li></ol> 4。トランスジェニックルートの識別<ol><li>二週間根粒菌接種後、複合植物は水で洗浄することにより、最大-根ざしている。根は容易に水に砂やパーライトから分離することができる。 </li><li>我々は、UV -顕微鏡（ニコンSMZ1500、励起460 - 500nmの、ダイクロイック505nm、510nm以上のバリア）の下に毛状根を置く。トランスジェニックのルーツは、GFP陽性であり、不定根はGFP陰性である。私たちは、その後、根accordinを収集するグラムGFPの信号、および、結節数をカウントルートの長さを測定し、側根の密度を計算する。 GFP陰性の根を対照として使用されています。 </li></ol> 5。代表結果私たちの実験では、新しい毛根はA.後2-3週間で外植片から再生するリゾゲネス innoculation。 UV -顕微鏡下で、GFP遺伝子を有するトランスジェニックルーツは強い緑色の蛍光（図1）を示す。再生ルーツとGFP陽性の根の部分の量は、外植片と複合plants.Onの平均の生育環境の条件に依存し、生成された根の25％が遺伝子組み換え毛状根3であった。形質転換効率、1）を高めるためにとしてビデオに表示されているプラ​​スチックのカバーの運命は、、最初の数日間のための成長のトレイの湿度を維持するのに十分であり、そして植物は最初の数日間で少し水を必要とする。過度の水遣りは、毛状根の形成に有害である、2） アグロバクテリウム innoculantの濃度は形質転換のために重要です。細胞の過剰な量は、毛状根の形成や結節形成に有用ではない。結節形成のため、散水ソリューションは窒素フリーにする必要がある、そうでなければ、いくつかの結節が形成されます。 毛状根複合植物を原料として子葉またはentactの苗を使用して生成することができます。上記のプロトコルのマイナーな修正だけでは、他の組織8から毛状根を生成するためにncessaryです。重要なのは、それぞれの毛状根は、独立した形質転換事象です。したがって、一つの複合プラントにおいて観察された表現型は、複数のコンポジット工場で繰り返しによって確認するために必要ないくつかのtransforamtionイベントの合計です<img src="/files/ftp_upload/2633/2633fig1.jpg" alt="図1" /> 図1。 A.トランスジェニックルーツは、毛状根からソートすることができます。 B.の結節は、毛状根複合植物に形成された。我々は、4週齢のMを配置するUV -顕微鏡とGFP陽性の根の下にtruncatula毛状根の植物は簡単に識別することができます。 （赤矢印：GFPのルート、黄色矢印：非GFPのルート） <table border="1"><tbody><tr><td>在庫切れ</td><td> g/200ml </td><td> mlストック/リットル</td></tr><tr><td>のMgSO 4 · 7H 2 O </td><td> 12.3 </td><td> 2 </td></tr><tr><td>のCaCl 2 · 2H 2 O </td><td> 14.7 </td><td> 4 </td></tr><tr><td> K 2 HPO 4 · 3H 2 O </td><td> 6.8 </td><td> 1 </td></tr><tr><td> K 2 SO 4 </td><td> 11 </td><td> 4 </td></tr><tr><td>鉄塩素3 · 6H 2 O </td><td> 0.49 </td><td> 2.5 </td></tr><tr><td>微量栄養素</td><td>下記参照してください。 </td><td> 1 </td></tr></tbody></table><table border="1"><tbody><tr><td>微量栄養素</td><td>リットル当たりのグラム</td></tr><tr><td> H 3 BO 3 </td><td> 0.142 </td></tr><tr><td> MnSO 4 H 2 O </td><td> 0.077 </td></tr><tr><td> ZnSO 4 · 7H 2 O </td><td> 0.1725 </td></tr><tr><td>のCuSO 4 .5 H 2 O </td><td> 0.037 </td></tr><tr><td> NaMoO 4。 H 2 O </td><td> 0.024 </td></tr><tr><td>のCoCl 2。 H 2 O </td><td> 0.0025 </td></tr><tr><td> NISO 4 </td><td> 0.001 </td></tr></tbody></table> 表1。Nitogenフリー栄養液<table border="1"><tbody><tr><td> K 2 HPO4 </td><td> 0.5グラム</td></tr><tr><td> NaClの</td><td> 0.1グラム</td></tr><tr><td>し、MgSO 4 · 7H 2 O </td><td> 0.2グラム</td></tr><tr><td>酵母エキス</td><td> 0.4グラム</td></tr><tr><td>マンニトール</td><td> 10グラム</td></tr><tr><td> pH値= 6.8 </td><td></td></tr></tbody></table> 表2。酵母はマンニトール培地を抽出（1リットル当たり）

<ol>
	<li>Limpens, E., Ramos, J., Franken, C., Raz, V., Compaan, B., Franssen, H., Bisseling, T., Geurts, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+in+Agrobacterium+rhizogenes-transformed+roots+of+Arabidopsis+and+Medicago+truncatula.">RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula.</a> J. Exp. Bot. 55, 983-992 (2004).</li><li>Collier, R., Fuchs, B., Walter, N., Kevin, L. W., Taylor, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vitro+composite+plants:+an+inexpensive,+rapid+method+for+root+biology.">Ex vitro composite plants: an inexpensive, rapid method for root biology.</a> Plant J. 43, 449-457 (2005).</li><li>Zhang, J., Subramanian, S., Stacey, G., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavones+and+flavonols+play+distinct+critical+roles+during+nodulation+of+Medicago+truncatula+by+Sinorhizobium+meliloti.">Flavones and flavonols play distinct critical roles during nodulation of Medicago truncatula by Sinorhizobium meliloti.</a> Plant J. 57, 171-183 (2009).</li><li>Boisson-Dernier, A., Chabaud, M., Garcia, F., Becard, G., Rosenberg, C., Barker, 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=Agrobacterium-rhizogenes-transformed+roots+of+Medicago+truncatula+for+the+study+of+nitrogen-fixing+and+endomycorrhizal+symbiotic+associations.">Agrobacterium-rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations.</a> Mol. Plant-Microbe Interact. 14, 695-700 (2001).</li><li>Zhang, J., Subramanian, S., Zhang, Y., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flavone+synthases+from+Medicago+truncatula+are+flavanone-2-hydroxylases+and+are+important+for+nodulation.">Flavone synthases from Medicago truncatula are flavanone-2-hydroxylases and are important for nodulation.</a> Plant Physiol. 144, 741-751 .</li><li>Subramanian, S., Hu, X., Lu, G., Odelland, J., Yu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+promoters+of+two+isoflavone+synthase+genes+respond+differentially+to+nodulation+and+defense+signals+in+transgenic+soybean+roots.">The promoters of two isoflavone synthase genes respond differentially to nodulation and defense signals in transgenic soybean roots.</a> Plant Mol Biol. 54, 623-639 (2004).</li><li>Vincent, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> A manual for the practical study of root nodule bacteria International Biological Program Handbook. , 1-13 (1970).</li><li>Subramanian, S., Graham, M. Y., Yu, O., Graham, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+Interference+of+Soybean+Isoflavone+Synthase+Genes+Leads+to+Silencing+in+Tissues+Distal+to+the+Transformation+Site+and+to+Enhanced+Susceptibility+to+Phytophthora+sojae.">RNA Interference of Soybean Isoflavone Synthase Genes Leads to Silencing in Tissues Distal to the Transformation Site and to Enhanced Susceptibility to Phytophthora sojae.</a> Plant Physiol. 137, 1345-1353 (2005).</li></ol>

利害の衝突は宣言されません。

毛状根の複合プラントを生成すると、多くの双子葉植物の種のトランスジェニック材料を大量に得るために迅速かつ容易な方法です。このメソッドは、トランスジェニック種子を生産することはできませんが、それは数週間でトランスジェニック材料を生成することができます。方法は困難組織培養の確立や、安定した形質転換体を生成している植物に特に適しています。長年にわたり、我々は、遺伝子機能、プロモーターの機能、マイクロRNA、根と側根の発達、防御と非生物的ストレス応答、根粒形成および他の共生のプロセス、ホルモン応答、メタボリックプロファイリング、遺伝子プロファイリング、プロテオーム解析の研究にこの技術を使用している、と他の生物学的プロセス。プロトコルは、堅牢で複製可能です。

generation of composite plants in medicago truncatula used for nodulation assays

アグロバクテリウムのtumerfaciensと同様に、アグロバクテリウムリゾゲネスは、自律的なルート誘導（Ri）のプラスミド。A.に基づいて植物細胞に外来DNAを転送することができますリゾゲネスは、植物組織やフォームの変換後の複合植物の毛状根の形成を引き起こす可能性があります。芽はまだ非トランスジェニックいる間、エネルギーや成長支援を提供するために提供する、これらの複合の植物で、再生された根の一部は、野生型T - DNAと人工バイナリーベクターを有するトランスジェニック、です。これらの毛状根の複合植物が遺伝子組み換え種子を生成しませんが、植物の研究でこれらの複合植物は非常に便利なように重要な機能がいくつかあります。最初に、広い宿主範囲を持つ、A.リゾゲネスは 、様々な種の遺伝子工学を可能にする、特に多くの植物種、双子葉植物を変換することができます。第二に、A.リゾゲネスは、直接組織し、外植片に感染、形質転換前には組織培養では手に負えない植物種の形質転換に最適です、複合植物を得るためには必要ありません。また、トランスジェニックルートの組織は、数週間のうちに生成することができます。 ウマゴヤシtruncatulaの場合、我々は正常な花のディップシロイヌナズナ形質転換よりも速く、短く3週間でトランスジェニックルーツを取得することができます。全体的に、毛状根の複合プラントテクノロジーは、遺伝子の機能とルートに関連する、表現型を研究するために汎用性と有用なツールです。ここでは、毛状根複合植物が困難な変換種M.の植物-根粒菌の相互作用と根粒形成を研究するために使用することができる方法を示していますtruncatula。

Watch this Scientific Journal Video about Generation of Composite Plants in Medicago truncatula used for Nodulation Assays at JoVE.com

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

私たちは、毛状根複合植物が困難な変換種で植物 - 根粒菌の相互作用と根粒形成を研究するために使用することができる方法を示しています<em&gt;ウマゴヤシtruncatula</em&gt;。

の複合植物の世代ウマゴヤシtruncatula根粒形成のアッセイに使用

Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) ...

generation-composite-plants-medicago-truncatula-used-for-nodulation

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Biology

Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

15.9K ビュー.  Donald Danforth Plant Science Center. 私たちは、毛状根複合植物が困難な変換種で植物 - 根粒菌の相互作用と根粒形成を研究するために使用することができる方法を示していますウマゴヤシtruncatula。

ビデオ: Generation of Composite Plants in Medicago truncatula used for Nodulation Assays

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical shock or electrical stimulation is available for study of various aspects of seizure activity and behavior. All flies, including wild-type, will undergo seizure-like activity if stimulated at a high enough voltage. Seizure like activity is an all-or-nothing response and each genotype has a specific seizure threshold. The seizure threshold of a specific genotype of fly can be altered either by treatment with a drug or by genetic suppression or enhancement. The threshold is easily measured by electrophysiology. Seizure-like activity can be induced via high frequency electrical stimulation delivered directly to the brain and recorded through the dorsal longitudinal muscles (DLMs) in the thorax. The DLMs are innervated by part of the giant fiber system. Starting with low voltage, high frequency stimulation, and subsequently raising the voltage in small increments, the seizure threshold for a single fly can be measured.

Neurocircuit Assays for Seizures in Epilepsy Mutants of Drosophila

Using high frequency electrical stimulation, seizure-like activity can be induced in Drosophila. This activity is easily recorded from the giant fiber system.

のてんかんの変異体における発作のためのアッセイをNeurocircuitショウジョウバエ</em

Drosophila melanogaster is a useful tool for studying seizure like activity. A variety of mutants in which seizures can be induced through either physical ...

生物学

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

使用シュワン-アストロサイト相互作用の解析インビトロアッセイ

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

植物の転写産物の正確な位置のためのin situハイブリダイゼーション

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

培養中の細胞のための生存率アッセイ

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

仙台ウイルスのヒト体細胞からの効率的な次世代ヒューマン誘導多能性幹細胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied1. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described.&#160;

代謝産物の調査のためのMALDI-質量分析イメージング<em&gt;ウマゴヤシtruncatula</em&gt;ルート結節

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

時空のCaを測定する<sup&gt; 2 +</supシロイヌナズナ植物における&gt;シグナル

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological samples, designed for high-resolution magnetic resonance imaging (MRI), also referred to as MR microscopy (MRM). It may be used at pre-clinical MRI spectrometers, demonstrated on Medicago truncatula root samples. Microcoils increase sensitivity by matching the size of the RF resonator to the size of the sample of interest, thereby enabling higher image resolutions in a given data acquisition time. Due to the relatively simple design, solenoidal microcoils are straightforward and cheap to construct and can be easily adapted to the sample requirements. Systematically, we explain how to calibrate new or home-built microcoils, using a reference solution. The calibration steps include: pulse power determination using a nutation curve; estimation of RF-field homogeneity; and calculating a volume-normalized signal-to-noise ratio (SNR) using standard pulse sequences. Important steps in sample preparation for small biological samples are discussed, as well as possible mitigating factors such as magnetic susceptibility differences. The applications of an optimized solenoid coil are demonstrated by high-resolution (13 x 13 x 13 &#956;m3, 2.2 pL) 3D imaging of a root sample.

MRM Microcoil Performance Calibration and Usage Demonstrated on Medicago truncatula Roots at 22 T

A protocol to study biological tissue at high spatial resolution using ultra-high field magnetic resonance microscopy (MRM) using microcoils is presented. Step-by-step instructions are provided for characterizing the microcoils. Finally, optimization of imaging is demonstrated on plant roots.

22 Tで 医薬トランカチュラ ルーツで実証MRMマイクロコイル性能キャリブレーションと使用

This protocol describes a signal-to-noise ratio (SNR) calibration and sample preparation method for solenoidal microcoils combined with biological ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

菌従属栄養植物組織における真菌のコロニー形成と種子の共生発芽を解釈するための顕微鏡技術

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...

AirID-Based Proximity Labeling for Protein-Protein Interaction in Plants

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA (known as BioID) have proven successful in identifying protein-protein interactions (PPIs). APEX, BioID, and TurboID, a revised version of BioID have some restrictions in addition to being valuable technologies. The recently developed AirID, a novel version of BioID for proximity identification in protein-protein interactions, overcame these restrictions. Previously, AirID has been used in animal models, while the current study demonstrates the use of AirID in plants, and the results confirmed that AirID performs better in plant systems as compared to other PL enzymes such as BioID and TurboID for protein labeling that are proximal to the target proteins. Here is a step-by-step protocol for identifying protein interaction partners using AT4G18020 (APRR2) protein as a model. The methods describe the construction of vector, the transformation of construct through agroinfiltration, biotin transformation, extraction of proteins, and enrichment of biotin-labeled proteins through affinity purification technique. The results conclude that AirID is a novel and ideal enzyme for analyzing PPIs in plants. The method can be applied to study other proteins in plants.

Here, we present a step-by-step protocol for performing the proximity labeling (PL) experiment in cucumber (Cucumis sativus L.) using AT4G18020 (APRR2)-AirID protein as a model. The method describes the construction of a vector, the transformation of a construct through agroinfiltration, biotin infiltration, protein extraction, and purification of biotin-labeled proteins through affinity purification technique.

植物におけるタンパク質間相互作用のためのAirIDベースの近接標識

In mammalian cells and plants, proximity labeling (PL) approaches using modified ascorbate peroxidase (APEX) or the Escherichia coli biotin ligase BirA ...