1. Growth of flax under inducing conditions.
<ol>
 <li>5" pots are filled with soil and firmed.</li>
 <li>Three seeds per pot are planted &frac14; inch deep and the soil firmed over the seeds.</li>
 <li>The pots are then watered with 100ml of the appropriate nutrient solutions
 <ol>
 <li>The non-inducing control (C) conditions use a 1/10th strength Miracle-Gro every week. The high nutrient condition (NPK treatment) had full strength Miracle Grow applied weekly. Low nutrient - plants only had tap water applied throughout their growth. (Durrant 1971, Cullis 1980).</li>
 </ol>
 </li>
 <li>The plants are grown under natural light during the summer. During the winter they are grown under sodium lights with a 16/8 hour light/dark cycle.</li>
 <li>At weekly intervals leaves and meristems are collected.</li>
 <li>The plant material is quick frozen in liquid nitrogen.</li>
</ol>
Results
The three genotypes grow at different relative rates depending on the conditions (Figure 1). Therefore under control conditions the Pl line grows best with L being more vigorous than S (Figure 1a). Under low nutrients (water), S grows better than either L or Pl (Figure 1b). Under high nutrient (NPK) L grows better than Pl which only grows slightly better than S (Figure 1c). A comparison between each of the lines grown under the three differing treatments is shown in Figure 1 d - f. Pl grows best under control conditions (Figure 1d), L best under high nutrient (Figure 1e) and S similarly under both high nutrient and control conditions (Figure 1f).
These data show that the three lines can be differentiated based on their growth under the three environments. Of note is that Pl grows best under the control conditions while L grows best under the NPK conditions (under which it was induced). S grows about the same in all three conditions, that is, it is not able to take advantage of improved nutrient conditions but is better able to grow under very low nutrient conditions.
The growth parameters that are relevant to the differentiation of the plants include the plant height (and associated with this the internode length, that is the distance between each leaf), the leaf size and the degree of branching. For Pl under control conditions the plant is tall with branches and the leaves are large and dark green. Under low nutrients the plant is very short, the distance between each leaf is also very short and the leaves are small and light green. The leaves at the tip are darker green than those lower down the stem. Under high nutrients the plant looks very similar to that under control conditions except that both the main stem and the side shoots are shorter than in the control plants. For the large genotroph the plants look very similar to those of Pl except that the most vigorous growth in under high nutrients. Again under low nutrients the plant is very short and has light green leaves. The S genotroph grows similarly in all three environments although the leaves are lighter green under the low nutrients. However, in the low nutrient conditions the S genotroph is much more vigorous than either of the Pl or L lines.
2. RNA and DNA extractions from flax meristems and leaves are done separately. The Roche total RNA prep kit was used for the RNA extractions and the Qiagen DNeasy Plant DNA prep kit was used for the DNA extractions.
RNA Extraction
<ol>
 <li>3 meristems plus some surrounding leaves are moved from storage cryovial into eppendorf tube and placed into liquid nitrogen until ready to be ground.</li>
 <li>Sterile sand is added to tube and tissue is ground using a micropestle until fine.</li>
 <li>400 ul lysis/binding buffer from Roche total RNA prep kit is added and tissue is further ground until no clumps are visible.</li>
 <li>Sample is vortexed for 15 sec to aid lysis and then spun for 1 min at 13,000 rpm to collect sand and any debris.</li>
 <li>Supernatant is added to spin column and the rest of the RNA prep kit instructions are followed as directed.</li>
</ol>
DNAse treatment
<ol>
 <li>1/10 RNA sample volume of 10X DNAse buffer is added.</li>
 <li>30 units of DNAse is added and reaction is incubated at 37&deg;C for 30 min and 70&deg;C for 5 min.</li>
</ol>
Reverse Transcription
<ol>
 <li>Applied Biosystems RNA to cDNA kit is used as per directions, reaction incubated at 37&deg;C for 1 hour and 95&deg;C for 5 minutes.</li>
</ol>
The cDNA is then used in PCR or for qPCR
qPCR
qPCR was performed using an ABI Step One system. All assays were performed in triplicate on each run. The actin gene was used as a copy number control as the best available housekeeping gene.
<ol>
 <li>The appropriate primer pair was added to each tube in 1&mu;l.</li>
 <li>The cDNA was diluted to the appropriate concentration and 9&mu;l added to each tube</li>
 <li>10&mu;l of the 2x Power SYBR Green PCR MasterMix (ABI) and was added to each tube</li>
 <li>The program amplification program was:
 <ul>
 <li>10 minutes at 95&deg;C</li>
 <li>40 cycles of 15sec at 95&deg;C, 1 min at 60&deg;C</li>
 </ul>
 
 Thermal denaturation step to verify amplification specificity</li>
 <li>The relative expression levels were determined by the value of 2(CTunknown - CTactin).</li>
</ol>
DNA preparation
<ol>
 <li>Buffer AP1 from Qiagen DNeasy Plant DNA prep kit is added to 6 leaves with sterile sand in a microfuge tube. The tissue is ground with micropestle until no clumps are visible.</li>
 <li>The kit instructions are followed as directed.</li>
</ol>
Results
PCR amplification from the DNA isolated from leaves at various positions along the stem demonstrates that the appearance of LIS-1 occurs while the plants are growing under inducing conditions (4). The qPCR results show that the presence of LIS-1 (or other genomic changes associated with the induction) affects the expression of genes in the immediate vicinity of LIS-1 (Figure 2). The six genes shown in Figure 2 are all differentially expressed in Pl and S with four having higher expression in PL (without LIS-1) and two having higher expression in S (which does have LIS-1). Panel 1 and 2 are the two genes closest to the insertion point of LIS-1. The expression of these gene s in the large genotroph (which has had changes induced but does not have LIS-1) and the expression under different growth conditions will be needed to determine any causal relationship between LIS-1 and the expression changes.
<img src="/files/ftp_upload/2332/2332fig1.jpg" alt="Figure 1" /> Figure 1. Pl, L and S grown under three different nutrient regimes. Panels a - control, b - low nutrients and c - NPK. d - Pl, e - S, f - L. Panels d - f from left to right: low nutrients, control and NPK. D - Pl, e - L, F - S.
<img src="/files/ftp_upload/2332/2332fig2tmb.jpg" alt="Figure 2 thumb" /> Figure 2. Gene expression around the point of insertion of LIS-1. Gene 1 (Inhibitor of growth) is immediately 5' to LIs1 and the gene 2 (Kip-related cyclin-dependent kinase inhibitor id 3" of LIS-1. The other genes are all along the same scaffold (~500kb) built from the complete flax genome sequence. Please <a href="https://www.jove.com/files/ftp_upload/2332/2332fig2.jpg">click here to see a larger figure</a>.
<img src="/files/ftp_upload/2332/2332fig3.jpg" alt="Figure 3" /> Figure 3. PCR amplifications of DNAs extracted from individual leaves of plants of Stormont Cirrus grown under low nutrient conditions. The PCR products were separated on a 2% agarose-TBE gel. The uninserted site is shown in Panel a, the two ends of the inserted site are shown in Panels b and c. Leaf samples 1 - 6 were isolated at weekly intervals with sample 1 being the earliest after sowing.

<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>

No conflicts of interest declared.

Flax appears to have an inherently unstable genome. A number of commercial fiber flax varieties rapidly become non-uniform when grown for a few generations. The same observations do not appear to hold true for the oil flax varieties. Therefore there appears to be an association between the selection for fiber quality and genome instability. The environmentally induced heritable changes in flax were originally identified through the investigation of the agronomic observations of the growth of the fiber crop. These repeatable observations of specific phenotypic and genomic variations raises the question of how environmental stress can influence the suite of genomic rearrangements that can occur. The repeated appearance of LIS-1 in response to particular environments is consistent with this mutation being directly or indirectly selected. The growth of the lines induced by various treatments also indicates an improved performance under the relevant inducing conditions. The changes in expression also associated with the presence of LIS-1 again indicates that this insertion affect plant performance. The video presentation provides a forum for observing the plant phenotypic changes in a manner impossible to convey through traditional printed material.

<ul>
<li>Flax seeds of the original Stormont Cirrus variety denoted Pl and the genotrophs L and S.</li>
<li>5" pots</li>
<li>potting soil</li>
<li>Miracle-Gro</li>
<li>greenhouse</li>
<li>High pressure sodium lights</li>
<li>Liquid nitrogen </li>
<li>Mortar and pestles</li>
<li>Micropestles and microfuge tubes</li>
<li>Thermocycler</li>
<li>Agarose gel electrophoresis equipment</li>
<li>Digital imaging setup</li>
<li>StepOne qPCR module</li>
<li>Qiagen DNeasy Plant DNA prep kit</li>
<li>Applied Biosystems RNA to cDNA kit</li>
<li>Roche total RNA prep kit</li>
</ul>

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.

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

Environmentally Induced Heritable Changes in Flax

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.

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 visualizações.  Case Western Reserve University. Crescendo algumas variedades de linho com resultados de tensão de nutrientes em variações genômicas dentro de um subconjunto do genoma e da variação fenotípica. A inserção complexo em um local específico está associado com o crescimento sob vários regimes de nutrientes e com mudanças na expressão gênica em torno deste site.

Vídeo: 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.

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 ...

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.

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.

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.

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.

Monitoring Equilibrium Changes in RNA Structure by &#039;Peroxidative&#039; and &#039;Oxidative&#039; Hydroxyl Radical Footprinting

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

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.

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.

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.

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.

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.

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