1。亚麻诱导条件下生长。 <ol><li> 5"盆用土填充和坚定。 </li><li>每锅三种子种植¼英寸深和土壤的种子落实。 </li><li>盆，然后浇水，适当的营养液百毫升<ol><li>非诱导对照组（C）条件下使用一个1 / 10的力量创造奇迹的格罗每星期。高营养状况（氮磷钾治疗）有充分的力量奇迹增长每星期。低营养 - 植物只有在其整个发展应用的自来水。 （达兰特1971年，库里斯1980年）。 </li></ol></li><li>自然光下的植物生长在夏季。他们在冬季种植与16 / 8小时，光/暗周期下钠灯。 </li><li>每周一班的叶和分生组织收集。 </li><li>植物材料在液氮速冻。 </li></ol>结果在3个基因型的生长在不同的条件（图1）的相对比率。因此，控制的条件下的PL线最好用L比S（图1a）的大力增长。在低养分（水），S成长优于L或PL（图1b）。在高养分（氮磷钾）长的增长比PL只增长比S（图1c）稍微好一点。三个不同的治疗下生长的每个线之间的比较显示在图1 D - F。 PL增长最好控制的条件下（图1d），高营养和控制条件下同样大号下最好的高营养（图1e）和S（图1F）。 这些数据表明，基于他们的三种环境下的生长，可以区别的三条线。值得注意的是，PL控制条件下生长最好而L增长最好的下氮磷钾条件（诱导）。在所有这三个条件，即它不能改善营养条件的优势，但在非常低营养条件下能够更好地成长，小号的增长大致相同。 有关植物的分化生长参数包括（间长度，即每一片叶子之间的距离）的株高，叶片大小和分支的程度。对于PL控制条件下的植物是用树枝高大，叶大和深绿色。在低营养的植物很短，每一片叶子之间的距离也很短，叶子是绿色体积小，重量轻。尖的叶子比那些低下来的干较暗的绿色。在高营养的植物看起来非常相似，控制的条件下，除了主茎和方竹笋比对照植株在短。对于大型genotroph的植物看起来非常相似的PL者，但最具活力的增长下的高营养。下再次低营养的植物很短，浅绿色的叶子。同样的S genotroph增长在所有三个环境虽然在低养分较轻的绿色叶片。然而，在低营养条件下的S genotroph是严厉得多的PL或L线之一。 2。 RNA和DNA的提取亚麻分生组织和叶分别进行。罗氏公司的总RNA制备试剂盒用于RNA的提取和Qiagen公司DNeasy植物DNA制备试剂盒的DNA提取使用。 RNA的提取<ol><li> 3分生组织中加上一些周边叶移动存储离心管，Eppendorf管中，并置于液氮，直至地面。 </li><li>添加到无菌砂管和地面使用，直到精细micropestle组织。 </li><li> 400 UL罗氏总RNA准备试剂盒的裂解/结合缓冲液中添加和组织进一步的地面，直到没有团块可见。 </li><li>样品振荡15秒，以帮助裂解和纺1分钟13,000转，收集砂和任何杂物。 </li><li>上清液加入离心柱，随后其余的RNA制备试剂盒说明书的指示。 </li></ol> DNA酶处理<ol><li> 10X DNA酶缓冲液的样品量的1 / 10的RNA。 </li><li> 30个单位的DNA酶是添加反应是在37℃孵育° C为30分钟，70℃5分钟。 </li></ol>反转录<ol><li>美国应用生物系统公司的RNA的cDNA试剂盒是用来为每方向，反应孵育在37℃，1小时和95 ° C为5分钟。 </li></ol>然后用PCR或为定量PCR的cDNA 定量PCR 定量PCR进行使用ABI步骤的一个系统。在每次运行时，所有检测一式三份。肌动蛋白基因被用来作为一个拷贝数控制最好的看家基因。 <ol><li>适当的引物被添加到每个试管中加入1μl。 </li><li>的cDNA稀释至适当浓度和9μl添加到每个管</li><li> 2x电源SYBR GREEN PCR反应试剂（ABI）的10μL，被添加到每个管</li><li>该计划扩增程序是: <ul><li> 10分钟在95 ° C </li><li> 15秒40的周期在95℃，在60℃1分钟</li></ul> 热变性步骤，以验证扩增的特异性</li><li>相对表达水平进行了测定值2（CTunknown - CTactin）。 </li></ol> DNA的制备<ol><li> Qiagen公司DNeasy植物DNA制备试剂盒缓冲AP1的是添加到6叶无菌砂在离心管。该组织是地面与micropestle，直到没有团块可见。 </li><li>按照试剂盒说明书。 </li></ol>结果从各个岗位沿干叶中分离出DNA PCR扩增表明，LIS - 1的外观发生，而植物的诱导条件下（4）发展。定量PCR结果显示，LIS - 1（或其他基因组相关的变化与感应）的存在会影响基因的表达在紧邻LIS - 1（图2）。在图2所示的六个基因差异表达的都是四份报告具有较高的表达在PL（不含​​LIS - 1）和2个具有较高的表达（确实有LIS - 1）在S PL和S。面板1和2 LIS - 1插入点最接近的两个基因。将需要在大genotroph（这已经改变而引起的，但没有LIS - 1）和不同的生长条件下的表达，这些基因的表达，以确定任何LIS - 1表达的变化之间的因果关系。 <img src="/files/ftp_upload/2332/2332fig1.jpg" alt="图1" /> 图1。PL，L和S三种不同的营养制度下成长。面板 - 控制，B - 低营养和C - 三元复合肥。 D - PL，E - S，F - 属面板ð - F从左至右:低营养，控制和NPK。 D - PL，E - L，F - S。 <img src="/files/ftp_upload/2332/2332fig2tmb.jpg" alt="图2拇指" /> 图2。LIS - 1插入点周围的基因表达。基因1（生长抑制剂）5&#39;立即LIs1和LIS - 1基因2（硖相关细胞周期蛋白依赖性激酶抑制剂ID 3"的其他基因，沿着相同的脚手架从内置（〜500KB）完整的亚麻基因组序列。请<a href="http://www.jove.com/files/ftp_upload/2332/2332fig2.jpg">点击这里查看大图</a> 。 <img src="/files/ftp_upload/2332/2332fig3.jpg" alt="图3" /> 图3:从个人的斯托蒙特Cirrus的低营养条件下生长的植物叶中提取的DNA的PCR扩增。 2％的琼脂糖TBE凝胶分离PCR产物。 uninserted网站事务委员会所示，两端插入网站显示面板B和C叶样品1 - 6被隔离在样品1，播种后最早每周一班。

<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>

亚麻出现，有一个内在的不稳定性的基因组。一些商业纤维亚麻品种迅速成为非均匀时为几代人成长。不会出现相同的看法持有真实的胡麻品种。因此，似乎是一个协会之间的纤维品质的选择和基因组不稳定。环境引起的遗传改变亚麻纤维作物生长的农艺性状观察的调查最初是通过确定。这些重复的意见，提出了具体的表型和基因变异的环境压力如何影响可能发生的基因重排的套件的问题。 LIS - 1在针对特定环境中重复出现这种突变直接或间接地选择是一致的。各种治疗引起的线条的增长也表明相关的诱导条件下提高性能。表达的变化，也再次LIS - 1的存在表明，此插入，影响植物的性能。视频演示，提供了一个观察植物的表型变化的方式不可能通过传统的印刷材料传达论坛。

<ul><li>亚麻籽原斯托蒙特Cirrus的各种表示PL的genotrophs L和S。 </li><li> 5"盆</li><li>盆栽土壤</li><li>创造奇迹的格罗</li><li>温室</li><li>高压钠灯</li><li>液氮</li><li>砂浆和杵</li><li> Micropestles和离心管</li><li>热循环</li><li>琼脂糖凝胶电泳设备</li><li>数码影像设置</li><li> StepOne扩增定量PCR模块</li><li> Qiagen公司DNeasy植物DNA制备试剂盒</li><li>美国应用生物系统公司的RNA的cDNA试剂盒</li><li>罗氏公司总RNA准备试剂盒</li></ul>

environmentally induced heritable changes in flax

一些亚麻品种，营养的压力，通过修改它们的基因组，这些修改可以通过许多代人继承。此外，这些基因组的变化相关的遗传表型变异1,2。三个不同的营养条件下生长的亚麻品种Cirrus的斯托蒙特（PL）可以保持诱导控制条件下，或成为稳定的修改，或高或低营养条件下的增长或大或小的genotroph分别。每个这些条件下，从最初的增长导致的线条出现在随后的几代人在同等条件下种植时长得更好，尤其是PL线生长最好的控制下，表明在高和低营养生长的植物正在治疗压力。与遗传变化的诱导有关的基因变化之一是插入元素的外观（LIS - 1）3，4，而植物营养应力下的增长。关于这个插入事件，亚麻品种Cirrus的斯托蒙特（PL）三个不同的营养条件下生长时可以保持不变（在控制条件下），插入出现在所有的植物在低营养和这个传播给下一代，或插入（或部分）的出现，但通过几代人（高营养）4传输。插入的外观的频率表明它积极的选择，这也与后代的生长反应一致。叶或分生组织在不同成长阶段的收获是用于DNA和RNA的分离。 RNA是用于确定在不同生长环境和/或T他在场的情况下/ LIS - 1的表达变化。隔离的DNA是用来识别那些发生在其中插入的植物。

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

Environmentally Induced Heritable Changes in Flax

一些亚麻品种在生长中的基因组变异的基因组和表型变异的一个子集，根据营养应力结果。一个复杂的插入是在一个特定的站点与各种营养制度下的生长和基因表达的变化，围绕本网站。

环境引起的遗传亚麻的变化

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 次观看.  Case Western Reserve University. 一些亚麻品种在生长中的基因组变异的基因组和表型变异的一个子集，根据营养应力结果。一个复杂的插入是在一个特定的站点与各种营养制度下的生长和基因表达的变化，围绕本网站。

视频: 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.

斑马鱼的响应行为的检测，以测量光强的变化

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 ...

科研

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.

双层弹性性能的监测变化的单分子方法

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.

变化在大鼠乳腺形态和患乳腺癌的风险

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.

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

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.

监测RNA结构平衡的变动情况"过氧化"和"氧化"羟基自由基足

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.

人类体细胞重编程为诱导多能干细胞（iPS细胞）用绿色荧光蛋白逆转录病毒载体

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.

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

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.

测量水润谷胱甘肽诱导饲养响应

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.

亚麻花香浸改造（<em&gt;亚麻</em&gt;）生成的转基因后代具有高转化率

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.

追踪药物诱导Colocalizational变化分析受体后内部贩运

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