1。誘導条件下での亜麻の成長。 <ol><li> 5&quot;ポットは、土壌を充填し、確定されています。 </li><li>ポットごとに3つの種子が植えられている¼インチ深いと土は、種子を介して確認した。 </li><li>ポットは、適切な栄養溶液100mlのと水遣りされています<ol><li>非誘導コントロール（C）の条件は、毎週1 / 10の強度ミラクル-グロを使用してください。高栄養状態（NPKの治療は）完全な強度のミラクルは毎週適用育てていた。低栄養 - 植物は彼らの成長全体に適用される水道水を持っていた。 （デュラン1971、カリス1980）。 </li></ol></li><li>植物は夏の間、自然光の下で栽培されています。冬の間、彼らは8分の16時間の明/暗サイクルでナトリウム灯の下で栽培されています。 </li><li>週間間隔で葉や分裂組織が収集されます。 </li><li>植物材料は、迅速に液体窒素中で凍結されています。 </li></ol>結果 3つの遺伝子型は、条件（図1）に応じて異なる相対的な速度で成長する。したがって、制御条件の下でのPLラインは、LはS（図1a）よりも活発であることで最高の成長。低栄養素（水）の下で、SはLまたはPL（図1b）のいずれかより良い成長。高栄養下で（NPK）、LはSよりわずかに良い成長（図1c）PLよりも優れて成長する。 F. - 3つの異なった治療法で育成した行のそれぞれの間の比較を図1のDに示されていますPLは高い栄養と制御条件（図1F）の両方で同様に高栄養（図1e）とSで最高の、Lを制御条件下で最高の成長（図1d）。 これらのデータは3行が3つの環境下での彼らの成長に基づいて区別できることを示している。注目すべきは、LはNPKの条件（それが誘導されたの下）の下で最高の成長ながら、PLは制御条件の下で最高の成長ということです。 Sが3つの条件すべてに同じ程度成長する、つまり、それは改善された栄養条件を活用することではありませんが、良い、非常に低栄養条件下で成長することが可能です。 植物の分化に関連する成長パラメータは、植物の高さ（とそれぞれの葉の間の距離ですこの節間長、関連付けられている）、葉の大きさや分岐の程度が含まれています。 plに関する制御条件の下で植物は枝と背が高いと葉が大きく、濃い緑色です。低栄養素の下で植物が非常に短い場合、各葉の間の距離も非常に短く、葉が小さく、薄い緑色です。先端の葉が茎ダウン低いものよりも暗い緑です。高い栄養素の下で植物は主茎や側枝の両方がコントロール植物に比べて短いことを除いて、制御条件の下でそれと非常によく似ています。大genotrophのための植物は高い栄養素の下で最も活発な成長点を除いてPLのものと非常によく似ています。再び低栄養素で植物は非常に短く、光の緑の葉を持っています。葉は低い栄養素の下に軽く緑色ですがS genotrophは、3種類の環境すべてで同じように成長する。しかし、低栄養状態でS genotrophはPLまたはLラインのどちらよりもはるかに活発です。 2。亜麻の分裂と葉からRNAとDNAの抽出を別々に行われます。ロシュトータルRNAプレップキットはRNA抽出のために使用され、キアゲンDNeasy植物DNAプレップキットは、DNA抽出に使用されていました。 RNA抽出<ol><li> 3分裂組織およびいくつかの周囲の葉は、エッペンドルフチュー​​ブにストレージクライオバイアルから移動し、地面になる準備ができるまで、液体窒素中に配置されます。 </li><li>滅菌砂をチューブに添加され、組織は罰金までmicropestleを使用して粉砕される。 </li><li>ロシュトータルRNAプレップキットから400 ulの溶解/結合バッファーを添加していない塊が表示されていないまで、組織はさらにグランドです。 </li><li>サンプルは、砂やごみを収集するために13,000 rpmで1分間スピンして溶解を助けるとに15のために秒をボルテックスしています。 </li><li>上清をカラムにスピンに追加され、指示されたとおりにRNAプレップキットの説明書の残りの部分が続いている。 </li></ol> DNase処理<ol><li> 10XのDNaseバッファーの1 / 10のRNAサンプルの量が追加されます。 </li><li> DNaseの30単位を添加し、反応を37℃でインキュベートされた℃で30分間、70℃で5分間のためのC。 </li></ol>逆転写<ol><li> cDNAをキットにアプライドバイオシステムズRNAが当たり方向、37度でインキュベート反応℃で1時間および95℃で5分間として使用されます。 </li></ol> cDNAは、PCRや定量PCRに使用されます定量PCR 定量PCRはABIのステップOneシステムを用いて行った。すべてのアッセイは、実行ごとに三連で行った。アクチン遺伝子はコピー数の制御などに使用された利用可能な最善のハウスキーピング遺伝子。 <ol><li>適切なプライマー対は、1μlの各チューブに添加した。 </li><li> cDNAは、適切な濃度と9μlに希釈し、各チューブに添加</li><li> 2倍のパワーSYBRグリーンPCRマスター（ABI）の10μlを、各管に添加した</li><li>プログラムの増幅プログラムがあった。 <ul><li> 95℃で10分° C </li><li> 95℃15秒の40サイクル° C、60 ° Cで1​​分</li></ul> 増幅の特異性を確認するために熱変性のステップ</li><li>相対的な発現レベルは2の値（ - CTactin CTunknown）により決定した。 </li></ol> DNA調製<ol><li>キアゲン社DNeasy植物DNAプレップキットからバッファAP1は、マイクロ遠心チューブに滅菌砂で6葉に追加されます。ない塊が表示されていないまで、組織はmicropestleで粉砕される。 </li><li>キットの指示は、指示どおりに続いている。 </li></ol>結果幹に沿って様々な位置での葉から単離したDNAからのPCR増幅は植物が誘導条件（4）の下で成長している間にLIS - 1の外観が発生したことを示しています。定量PCRの結果は、LIS - 1（または誘導に関連する他のゲノムの変化）の存在が、LIS - 1のすぐ近く（図2）の遺伝子の発現に影響を​​及ぼすことを示している。図2に示す6つの遺伝子は、すべての差動4つ以上のPLの発現（LIS - 1を適用していない）とSのより高い発現を（LIS - 1を持っていない）有する二つを持って使用して、PLとSで表されます。パネル1と2は、LIS - 1の挿入ポイントに近い位置にある2つの遺伝子である。大genotrophでこれらの遺伝子の発現（これは誘導される変化があったがLIS - 1を持っていない）とは異なる成長条件での発現は、LIS - 1と発現の変化との間に因果関係を判断するために必要となる。 <img src="/files/ftp_upload/2332/2332fig1.jpg" alt="図1" /> 図1。PL、LとSは、3種類の栄養素体制の下で成長した。パネル - コントロール、B - 低栄養素およびc - NPK。 D - PL、E - S、F - D L.パネル - fを左から右へ：低栄養素、コントロールとNPK。 D - PL、電子 - L、F - S. <img src="/files/ftp_upload/2332/2332fig2tmb.jpg" alt="図2親指" /> LIS - 1の挿入ポイントの周りの図2。遺伝子発現。遺伝子1（成長の阻害剤）LIS1にすぐに5&#39;であり、LIS - 1の遺伝子2（キップ関連のサイクリン依存性キナーゼ阻害剤のID 3&quot;。他の遺伝子はすべてから構築された同一の足場（〜500KB）に沿っている亜麻のゲノム配列を完了してください。下さい<a href="http://www.jove.com/files/ftp_upload/2332/2332fig2.jpg">大きな図をご覧いただくにはこちらをクリック</a> 。 <img src="/files/ftp_upload/2332/2332fig3.jpg" alt="図3" /> 図3。低栄養条件下で栽培ストーモントシーラスの植物の個々の葉から抽出したDNAのPCR増幅。 PCR産物を2％アガロース- TBEゲル上で分離した。 uninsertedサイトがパネルに示されている、挿入部位の両端は、パネルBおよびCに示されています葉のサンプル1から6は、サンプル1は、播種後早いいると毎週の間隔で分離した。

<ol>
	<li>Durrant, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+environmental+induction+of+heritablel+change+in+Linum.">The environmental induction of heritablel change in Linum.</a> Heredity. 17, 27-61 (1962).</li><li>Cullis, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+aspects+of+the+environmental+induction+of+heritable+changes+in+flax.">Molecular aspects of the environmental induction of heritable changes in flax.</a> Heredity. 38, 129-154 (1977).</li><li>Chen, Y., Schneeberger, R. G., Cullis, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+site-specific+insertion+sequence+in+flax+genotrophs+induced+by+the+environment.+New+Phytol.">A site-specific insertion sequence in flax genotrophs induced by the environment. New Phytol.</a> , 167-171 (2005).</li><li>Chen, Y., Lowenfeld, R., Cullis, C. 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+environmentally+induced+adaptive+(?)+insertion+event+in+flax.">An environmentally induced adaptive (?) insertion event in flax.</a> Int. J. Genet. Mol. Biol. 1, 38-47 (2009).</li></ol>

利害の衝突は宣言されません。

フラックスは、本質的に不安定なゲノムを持っているように見えます。数世代にわたり増殖させた場合の商用繊維亜麻の種の数は急速に不均一になる。同じ観察結果は、石油の亜麻の種のために真のまま表示されません。したがって、繊維の質とゲノム不安定性のための選択の間に関連があるように見える。亜麻の環境誘発遺伝性の変化は、もともと繊維作物の成長の農業観測の調査によって確認された。特定の表現型とゲノムの変化のこれらの再現性の観察結果は、環境ストレスが発生する可能性がゲノム再編成のスイートに影響を与えることができるかという疑問が生じる。特定の環境に応じて、LIS - 1の繰り返し出現は、この変異が直接または間接的に選択されていると一致している。様々な処理により誘導される行の成長はまた、関連する誘導条件下でのパフォーマンスの向上を示している。また、LIS - 1の存在に関連した発現の変化は、再びこの挿入は、プラントのパフォーマンスに影響を与えることを示しています。ビデオプレゼンテーションは、従来の印刷物を通じて伝えることが不可能な方法で植物の表現型の変化を観察するためのフォーラムを提供しています。

<ul><li>オリジナルストーモントシーラス品種の亜麻の種子は、PLとgenotrophs LとSを表す</li><li> 5&quot;ポット</li><li>土壌をポッティング</li><li>ミラクル-グロ</li><li>温室効果</li><li>高圧ナトリウム灯</li><li>液体窒素</li><li>モルタルと乳棒</li><li> Micropestlesおよびマイクロチューブ</li><li>クラー</li><li>アガロースゲル電気泳動装置</li><li>デジタルイメージングセットアップ</li><li> StepOne定量PCRモジュール</li><li>キアゲン社DNeasy植物DNAプレップキット</li><li> cDNAをキットにアプライドバイオシステムズRNA </li><li>ロシュトータルRNAプレップキット</li></ul>

environmentally induced heritable changes in flax

いくつか亜麻の品種は、そのゲノムを変更することによって、栄養ストレスに応答し、これらの変更は、多くの世代にわたって継承することができます。また、これらのゲノムの変化に関連付けられていると表現型のバリエーションの1,2を遺伝している。つの異なる栄養条件下で栽培亜麻多様ストーモントシーラス（PL）のどちらか（対照条件下）誘導性のまま、または安定的にそれぞれの高または低栄養条件下での成長が大きいか小さいgenotrophのいずれかに変更になることができます。これらの各条件の下で初期成長に起因する線は、PLラインが両方ともハイとローの栄養素で植物栽培が下にあることを示す制御処理で最高の成長著しく、その後の世代で同じ条件下で増殖した場合より良い成長に見えるストレス。植物が栄養ストレス下で成長している間に遺伝性変化の誘導に関連付けられているゲノムの変化の一つは、挿入要素の外観（LIS - 1）3、4です。この挿入イベントに関しては、つの異なる栄養条件下で栽培亜麻多様ストーモントシーラス（PL）のどちらか、（制御条件の下で）変更されないまま挿入はすべての植物に表示される（低栄養素の下）と、これが送信されていることができます次の世代に、または挿入（またはその一部）を持って表示されますが、世代（高い栄養素下）4を介して送信することはできません。この挿入の出現頻度は、それはまた次の世代の成長の応答と一致している正の選択、下にあることを示しています。成長の様々な段階で収穫された葉や分裂は、DNAとRNAの分離に使用されます。 RNAは、様々な成長の環境および/またはt彼LIS - 1の存在/不在に関連付けられている式の中で変化を識別するために使用されます。単離されたDNAは、挿入が発生しているそれらの植物を識別するために使用されます。

Watch this Scientific Journal Video about Environmentally Induced Heritable Changes in Flax at JoVE.com

Environmentally Induced Heritable Changes in Flax

ゲノムと表現型変異のサブセット内にゲノム変異の栄養ストレスの結果の下にある亜麻の品種を育てる。特定のサイトでの複雑な挿入は、様々な栄養素体制の下で成長したとこのサイトの周り遺伝子発現の変化と関連付けられています。

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 ...

environmentally-induced-heritable-changes-in-flax

Research

JoVE Journal

Biology

10.2K ビュー.  Case Western Reserve University. ゲノムと表現型変異のサブセット内にゲノム変異の栄養ストレスの結果の下にある亜麻の品種を育てる。特定のサイトでの複雑な挿入は、様々な栄養素体制の下で成長したとこのサイトの周り遺伝子発現の変化と関連付けられています。

ビデオ: Environmentally Induced Heritable Changes in Flax

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

光強度の変化にゼブラフィッシュの応答性を測定する行動アッセイ

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

生物学

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

二層弾性特性の変化をモニタリングするための単一分子法

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

ラットにおける乳腺の形態と乳癌リスクの変化

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

遺伝的にコードされたカルシウムのセンサを用いたアポトーシス時のミトコンドリア内カルシウム濃度の動的変更の監視

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling a specific biologic role as regulator, binder or catalyst. Information about tertiary contact formation is essential to understand the function of RNA molecules. Hydroxyl radicals (&bull;OH) are unique probes of the structure of nucleic acids due to their high reactivity and small size.1 When used as a footprinting probe, hydroxyl radicals map the solvent accessible surface of the phosphodiester backbone of DNA1 and RNA2 with as fine as single nucleotide resolution. Hydroxyl radical footprinting can be used to identify the nucleotides within an intermolecular contact surface, e.g. in DNA-protein1 and RNA-protein complexes. Equilibrium3 and kinetic4 transitions can be determined by conducting hydroxyl radical footprinting as a function of a solution variable or time, respectively. A key feature of footprinting is that limited exposure to the probe (e.g., 'single-hit kinetics') results in the uniform sampling of each nucleotide of the polymer.5
In this video article, we use the P4-P6 domain of the Tetrahymena ribozyme to illustrate RNA sample preparation and the determination of a Mg(II)-mediated folding isotherms. We describe the use of the well known hydroxyl radical footprinting protocol that requires H2O2 (we call this the 'peroxidative' protocol) and a valuable, but not widely known, alternative that uses naturally dissolved O2 (we call this the 'oxidative' protocol). An overview of the data reduction, transformation and analysis procedures is presented.

This protocol describes how to quantify the Mg(II)-dependent formation of RNA tertiary structure by two methods of hydroxyl radical footprinting.

&quot;過酸化&quot;と&quot;酸化&quot;ヒドロキシルラジカルフットプリントにより、RNAの構造における均衡の変化を監視する

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

GFPをレトロウイルスベクターを用いた誘導多能性幹細胞（性IPSC）にヒト体細胞を再プログラミングする

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

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 ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

ハイドラ内グルタチオンによって誘発される摂食応答を測定する

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

亜麻の花-DIP変換（<em&gt;アマ</em&gt;）高転換率のトランスジェニック子孫を生成するために、

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

Tracking Drug-induced Changes in Receptor Post-internalization Trafficking by Colocalizational Analysis

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and overall cell responsiveness to ligands and is, itself, influenced by intra- and extracellular conditions, including ligand-induced signaling. Optimized for use with monolayer-plated cultured cells, but extendable to free-floating tissue slices, this protocol uses immunolabelling and colocalizational analysis to track changes in intracellular receptor trafficking following both chronic/prolonged and acute interventions, including exogenous drug treatment. After drug treatment, cells are double-immunolabelled for the receptor and for markers for the intracellular compartments of interest. Sequential confocal microscopy is then used to capture two-channel photomicrographs of individual cells, which are subjected to computerized colocalizational analysis to yield quantitative colocalization scores. These scores are normalized to permit pooling of independent replicates prior to statistical analysis. Representative photomicrographs may also be processed to generate illustrative figures. Here, we describe a powerful and flexible technique for quantitatively assessing induced receptor trafficking.

Receptor trafficking modulates signaling and cell responsiveness to ligands and is, itself, responsive to cell conditions, including ligand-induced signaling. Here, we describe a powerful and flexible technique for quantitatively assessing drug-induced receptor trafficking using immunolabeling and colocalizational analysis.

Colocalizational解析による受容体後の内在トラフィッキングにおける薬物誘発性の変更の追跡

The intracellular trafficking of receptors is a collection of complex and highly controlled processes. Receptor trafficking modulates signaling and ...