Salaries and research support were provided by the United Soybean Board, and by State and Federal funds appropriated to The Ohio State University/Ohio Agricultural Research and Development Center. This research was also partially supported by a fellowship from CONACYT, Mexico, to CMHG. Mention of trademark or proprietary products does not constitute a guarantee or warranty of the product by OSU/OARDC and also does not imply approval to the exclusion of other products that may also be suitable. Journal Article No HCS 09-17.

The following methodology outlines a protocol for image analysis of gene expression using an automated image collection system. For ease of explanation, the overall approach was broken down into four steps: 1) seed preparation, 2) gene introduction using particle bombardment, 3) robotic image collection, and 4) image analysis. Although this general methodology can be used for a broad-range of other applications, the present sets of protocols are based on the use of the green fluorescent protein (gfp) gene, which is a useful reporter gene for tracking gene expression in the same piece of living tissue over time.
I. Seed Preparation
<ol>
	<li>Lima bean (Phaseolus lunatus cv. Henderson-Bush) seeds can be purchased, or ideally harvested from plants grown in a growth chamber (50% relative humidity, 16/8 h light:dark, 25/23&deg;C day/night). Post-harvest seed quality may be maintained by storing seeds at -20&deg;C.</li>
	<li>Prepare Magenta GA7 containers for germinating seeds. Fold filter paper or paper towels to fit in the bottom of Magenta boxes. Add ~25 ml deionized water to each container to moisten paper towels, and pour off unabsorbed water. Autoclave moistened paper-containing boxes for 20 min.</li>
	<li>Using 50 ml disposable centrifuge tubes, sterilize seeds in a 10% commercial bleach solution (20 seeds in 20-40 ml) for 20 min with shaking on a gyratory shaker at 60 rpm. 
 Note: All of the subsequent steps should be performed in a laminar flow hood.</li>
	<li>Rinse seeds 4-7 times with sterile deionized water with gentle agitation for 30 sec during each rinse. </li>
	<li>Place 5-6 sterile seeds between layers of folded paper located in each sterile Magenta box.</li>
	<li>Incubate seeds for 4 days under the following conditions: 25&deg;C, 16/8 h light:dark and 40 &mu;Em-2 s-1.</li>
</ol>
II. Gene Introduction using Particle Bombardment
<ol>
	<li>Build gene constructs using your favorite expression vector with a reporter gene that can be monitored in living tissues. We have engineered a high copy number expression vector useful for promoter and promoter element analyses. This vector contains the gfp gene which is used to visualize promoter/promoter element function in transformed tissues. </li>
	<li>Around 1-2 h before bombardment, excise lima bean cotyledons from germinating seedlings and remove the seed coats. Cotyledons suitable for bombardment should be yellow to light green, flat and free of any damage that might interfere with further image analysis.</li>
	<li>Place cotyledons on OMS culture medium containing MS salts1, B5 vitamins2, 3% sucrose and 0.2% Gelrite (pH 5.7) immediately after being excised.</li>
	<li>Precipitate the DNA construct onto M10 tungsten particles (Sylvania, Towanda, PA, USA). In a 0.6 ml microfuge tube, add 25 &mu;l tungsten particles (particles are resuspended in sterile water, 100 mg ml-1, right before use), 5 &mu;l DNA (1 &mu;g &mu;l-1), 25 &mu;l 2.5 M calcium chloride (Sigma-Aldrich Cat. C3881-500G) and 10 &mu;l 100 mM spermidine (Sigma Cat. S-2626). Vortex briefly to thoroughly mix all the components.</li>
	<li>Incubate the DNA preparation on ice for 5 min. Then, remove and discard 50 &mu;l supernatant.</li>
	<li>Resuspend the DNA-coated particles by vortexing and immediately remove a 2 &mu;l aliquot. Repeat this vortexing step every time an aliquot is removed from the tube. Coated particles should be kept in ice and used within 15 min.</li>
	<li>Through the top of a syringe filter, place 2 &mu;l coated particles in the middle of the filter screen. Place the filter unit containing the coated particles in the filter holding unit inside the particle gun chamber.</li>
	<li>Place a lima bean cotyledon adaxial side up on a baffle and place the baffle in the particle gun chamber. The baffle, which consists of a screen melted to the bottom of a beaker, is used as a platform to support tissues during bombardment.</li>
	<li>Bombard the tissues using a Particle Gun (in this methodology, we use a simple and inexpensive Particle Inflow Gun3, PIG, designed in our laboratory) as follows; a) open the valve leading to the vacuum to evacuate the chamber, b) after the vacuum reaches 760 mm (30 in) Hg, activate the solenoid and release the helium to propel the particles, c) after particle bombardment, close the vacuum line valve and release the vacuum using the exhaust valve. The helium pressure used to accelerate particles is 50 PSI.</li>
	<li>After the vacuum is released, open the chamber door to retrieve the bombarded cotyledons and return the cotyledon, adaxial side up, to OMS culture medium. Note: For quantification and expression profiling, we usually bombard a minimum of three cotyledons for every DNA construct. A positive control, which is usually a DNA construct that gives a well-studied gene expression profile, is included as a reference.</li>
</ol>
III. Automated Image Collection
<ol>
	<li>Turn on the mercury lamp and wait for 30 min for the lamp to warm up.</li>
	<li>Turn on the power sources for the Spot-RT CCD camera (Diagnostic Instruments Inc., Sterling Heights, MI, USA) and the robotics platform motor controller, which drives the movement of the robotics platform.</li>
	<li>Set the filter set for GFP detection (GFP2 filter set; Ex. 480/40 nm, Em. 510 LP) on a MZFLIII dissecting microscope (Leica, Heerbrugg, Switzerland).</li>
	<li>Sterilize the thickened polycarbonate Petri dish lids by spraying with 70% ethanol. The specialized lids, used to cover the Petri dish base prevent condensation during image collection4.</li>
	<li>Place the plates containing bombarded cotyledons on the robotics platform (Arrick Robotics, Hurst, TX, USA) of the automated image collection system. The robotics platform is driven by a stepping motor set and consists of a table for attachment of 8 Petri dishes. The platform is constructed of polycarbonate, polypropylene and aluminum. Plates are fastened in position by tightening a lateral plastic set-screw. </li>
	<li>Open the custom software application that controls both the robotics platform and image acquisition. Also, open the software white light controller switch.</li>
	<li>In the "motor motion" tab, click on "motor on" and "motor home". After the software orients the platform to the home position, we are ready to start entering positions for every cotyledon.</li>
	<li>In the "motor motion" tab, select the first plate. The platform will position the center of the Petri dish under the objective of the microscope. Using the moving distance buttons, precisely position the first cotyledon for image collection. With the white light controller on, the target region can be observed through the computer monitor by turning on the "live mode" feature.</li>
	<li>For the first cotyledon, set the focus and magnification (1.6x magnification is preferred) using the manual knobs on the dissecting microscope. Focus adjustments for the remaining cotyledons on the same plate are made using three leveling screws located below each plate, on the robotics platform.</li>
	<li>Once the region of interest has been set, click the "add this position" button. The software will add the coordinates of this region to a position schedule file and the platform will return to the center of the plate.</li>
	<li>Set positions and focus for all the remaining cotyledons on plates using the moving distance buttons and manual leveling screws. After all the coordinates of all the cotyledons have been entered, save the position schedule coordinates.</li>
	<li>Enter the image collection parameters. In the "image setting" tab, select the type of light desired; white or blue. For GFP detection, only the blue light should be used. Also, enter the exposure setting. Although we can modify time of exposure independently for red, blue and green color, we typically use the same settings for GFP photo-documentation (20, 14 and 14 sec for red, green and blue channels, respectively) allowing us to make direct and consistent comparisons among different DNA constructs.</li>
	<li>In the same "image setting" tab, specify an empty folder where the images will be stored. Every series of images will be saved within this master folder in independent folders, numbered sequentially according to the order of positions entered for cotyledons.</li>
	<li>Go to the "capture time control" tab and set the image acquisition time interval and total number of cycles of image collection. Images are typically collected every hour for 100 h, generating well-defined gene expression profiles.</li>
	<li>Start image acquisition by clicking on the "run schedule!" button located in the "position schedule" tab.</li>
	<li>At the end of the 100 h image collection, transfer all the images from the computer controlling the robot to any large capacity hard drive or computer for further image analysis. High-resolution images (1600 x 1200 pixels) are collected as TIF files measuring ~5 Mb each.</li>
</ol>
IV. Image Analysis
<ol>
	<li>Assemble the 100 sequential images from each folder using Adobe ImageReady.
	<ol>
 	<li>Open ImageReady and import all the sequential images composing a series as frames.</li>
	<li>Resize original images to 800 x 600 pixels. Collected images are high resolution, which generates large files which can make image processing slow.</li>
	<li>Scroll through the images and find a spot(s) visible in most images.</li>
	<li>Zoom up to 300% on the selected spot(s). Alignment of images using higher magnification allows for a more precise registration.</li>
	<li>Insert a layer on top of all images and make sure is visible in all frames. Mark the spot(s) location using the "paintbrush" tool.</li>
	<li>Start aligning all frames using the "move" tool and taking the marks on the layer as a reference.</li>
	<li>When alignment is complete, delete the layer used as a reference and save images as a single "psd" file. Also, export all the frames as a single "mov" file using the highest resolution. Manual alignment of each image series takes ~10 min.</li>
 </ol>
 </li>
	<li>Perform quantification of gene expression using ImageJ.
 <ol>
 	<li>Open ImageJ software and open the "mov" file saved previously in ImageReady.</li>
 	<li>Using the select tool, choose a 400 x 300 pixel area and crop the image. This new file should be saved as an "avi" file.</li>
 	<li>Separate the sequential images, located in the new "avi" file into red, blue and green channels by clicking "image", "color" and "RGB split" in ImageJ.</li>
 	<li>Subtract the background fluorescence from all images in the green and red channels. Using the green channel image series, select a 20 x 20 pixel area from a region with non-expressing cells and record its location in the "ROI Manager" tool, so that the same area will be selected in both channels. With the 20 x 20 pixel square active in the green channel, go to the "plugins" command located in the task bar of ImageJ and then click on the "subtract measured ROI for each slice" plugin-tool.</li>
 	<li>In ImageJ, click on "Image", "adjust" and then "threshold" to define or segment the GFP-expressing pixels. Adjust the threshold levels by dragging the bar in the threshold window to obtain an average spot size of 20-30 pixels.</li>
 	<li>Determine GFP expression using a plugin that we designed to measure the mean grayscale value per pixel and the total number of GFP-expressing pixels.</li>
 	<li>Copy the mean output grayscale values in ImageJ and paste into Microsoft Office Excel. Calculate the GFP expression for each channel (red and green) by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels for each channel. The sum of expression values for both channels gives the GFP expression. Values can be used to illustrate gene expression profiles and make direct comparisons. Time-lapse animations can be also generated using "mov" or "avi" files, providing a unique and clear visualization of the gene expression profile.</li>
</ol>
</li>
</ol>
V. Representative Results
The robotic image collection and analysis procedure (Fig. 1) reported here allows the acquisition of a large amount of quantitative data on gene expression in a short time period. For plant promoter characterization using the gfp gene, this methodology is not only useful to create transient expression profiles (Fig. 2) but also to track in a detailed-manner GFP expression in transiently- and stably-transformed plant tissues5,6. Short time-lapse animations generated with the collected sequential images are a valuable tool for in-depth analyses of gene expression over time in plant tissues. This methodology also has great application to evaluate factors that directly affect gene expression. For example, transient GFP expression under the presence of different suppressors of silencing of viral origin has been successfully studied using our automated image collection and analysis system7,8.
<img src="/files/ftp_upload/1733/1733fig1.jpg" alt="FIgure 1" /> Figure 1. Image analysis procedure consists of four main steps. (A) Acquisition of image series using the robotic image collection system, (B) separation of images into red, blue and green channels, (C) segmentation of the expressing pixels by adjusting the threshold levels, and (D) obtaining the output results containing the grayscale values and the GFP-expressing focus counting.
<img src="/files/ftp_upload/1733/1733fig2.jpg" alt="FIgure 2" /> Figure 2. Graph showing different transient expression profiles driven by plant promoters fused to gfp. Data was collected using our robotic image collection system and analyzed with ImageReady and ImageJ software.

<ol>
	<li>Murashige, T., Skoog, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+revised+medium+for+rapid+growth+and+bioassays+with+tobacco+tissue+cultures.">A revised medium for rapid growth and bioassays with tobacco tissue cultures.</a> Physiol Plant. 15, 473-497 (1962).</li><li>Gamborg, O. L., Miller, R. A., Ojima, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutrient+requirements+of+suspension+cultures+of+soybean+root+cells.">Nutrient requirements of suspension cultures of soybean root cells.</a> Exp Cell Res. 50, 150-158 (1968).</li><li>Finer, J. J., Vain, P., Jones, M. W., McMullen, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+particle+inflow+gun+for+DNA+delivery+to+plant+cells.">Development of the particle inflow gun for DNA delivery to plant cells.</a> Plant Cell Rep. 11, 232-238 (1992).</li><li>Finer, J. E., Finer, J. 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+simple+method+for+reducing+moisture+condensation+on+Petri+dish+lids.">A simple method for reducing moisture condensation on Petri dish lids.</a> Plant Cell Tiss Org. 91, 299-304 (2007).</li><li>Buenrostro-Nava, M. T., Ling, P. P., Finer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+35S+and+Lectin+promoters+in+transgenic+soybean+tissue+using+an+automated+image+acquisition+system+and+image+analysis.">Comparative analysis of 35S and Lectin promoters in transgenic soybean tissue using an automated image acquisition system and image analysis.</a> Plant Cell Rep. 25, 290-296 (2006).</li><li>Chiera, J. M., Bouchard, R. A., Dorsey, S. L., Park, E. H., Buenrostro-Nava, M. T., Ling, P. P., Finer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+two+highly+active+soybean+(Glycine+max+(L.)+Merr.)+promoters+and+their+characterization+using+a+new+automated+image+collection+and+analysis+system.">Isolation of two highly active soybean (Glycine max (L.) Merr.) promoters and their characterization using a new automated image collection and analysis system.</a> Plant Cell Rep. 26, 1501-1509 (2007).</li><li>Chiera, J. M., Lindbo, J. A., Finer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+and+extension+of+transient+GFP+expression+by+the+co-introduction+of+a+suppressor+of+silencing.">Quantification and extension of transient GFP expression by the co-introduction of a suppressor of silencing.</a> Transgenic Res. 17, 1143-1154 (2008).</li><li>Dhillon, T., Chiera, J. M., Lindbo, J. A., Finer, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+evaluation+of+six+different+viral+suppressors+of+silencing+using+image+analysis+of+transient+GFP+expression.">Quantitative evaluation of six different viral suppressors of silencing using image analysis of transient GFP expression.</a> Plant Cell Rep. 28, 639-647 (2009).</li></ol>

The use of robotics has enormous applications in different aspects of human life; specifically, robots have been effectively used to perform activities in dangerous environments, to automate tedious and complex activities, and to carry out tasks in a more precise way. In molecular biology, and specifically gene expression analyses, robots can help track not only features of genes but also tissue growth and development over time. Many biological phenomena occur dynamically, which may be difficult to follow using single time point observations.
The use of the gfp gene for expression studies brings additional advantages for observing tissue response and growth. For our experimental procedures, GFP allows us to follow gene expression in the same piece of tissue over time as GFP detection is a non-destructive. Furthermore, our version of the GFP protein is sufficiently stable to allow detection but also shows some turnover to minimize accumulation in plant tissues, allowing us to follow both the rise and fall of gene expression. 
We have already utilized our robotic image collection and analysis system for a wide range of applications. We foresee high potential for many biological applications where a dynamic understanding of a phenomenon is desired. For example, growth and development of plant tissues can be tracked using our system giving valuable insight/information on these processes. Also, the dynamics of protein transport using reporter genes like gfp, can be easily visualized using time-lapse animations. The methodology described in this report is technically complex but conceptually simple. Our results are robust and new applications are continually being discovered.

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robotics and dynamic image analysis for studies of gene expression in plant tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

Watch this Scientific Journal Video about Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues at JoVE.com

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

robotics-dynamic-image-analysis-for-studies-gene-expression-plant

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Biology

12.5K vistas.  The Ohio State University. Se presenta un método para la introducción, el seguimiento y el análisis cuantitativo de la expresión de GFP en las células vegetales. Este método utiliza un sistema diseñado a la robótica para la colección de imágenes semi-permanente de un gran número de muestras, con el tiempo. También demostrar el uso de ImageJ e ImageReady para el análisis de series de imágenes.

Video: Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...