In this procedure, the dynamics of gene expression in plant tissues are imaged over time with the use of robotics, first lima bean seeds are sterilized and placed between moistened paper towels in a tissue culture container for germination. After four days, the coan are removed and bombarded with a DNA construct containing a promoter fused to GFP Coan placed in Petri dishes are mounted on a two-dimensional custom designed robotics image collection system. The robot is programmed to capture images of each tissue piece every hour for 100 hours.
Images are subjected to image analysis, teed quantitative data on the intensity of GFP expression over time. Hi, I am John Finer, the director of the Plant Transformation Laboratory in the Department of Horticulture and Crop Science at the Ohio State University. I'm Carlos Herandez Garcia, PhD student in the final laboratory at the Ohio State University.
Today Carlos will show you the protocol that we developed for analysis of gene expression and plant tissues using the green fluorescent protein. We use this procedure that we have developed in the laboratory for measuring the strain of OD activity. To do this promoter our first crown upstream of GFP gene, which is our reporter genes.
I will show you how we introduce the promoter control into Lima vein Cataly and send an app on our custom design robotic image capture system. I will finish by going over our protocol for image analysis. So let's go To begin this procedure surface sterilized lima bean seeds using standard procedures.
Place the sterilized seeds in magenta GA seven containers between layers of moist and paper towels and incubate them for four days at 25 degrees Celsius. Here we use 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 or promoter element function in transformed tissues around one to two hours before bombardment.
Excise lima bean CU leadin from seed coats. CU leadin suitable for bombardment should be yellow to light green flat and free of any damage that might interfere with further image analysis immediately after excision placed 12 cu leadin in a Petri dish with OMS culture medium containing MS salts B five vitamins, 3%sucrose and 0.2%gel rate at pH 5.7. Next, precipitate the DNA construct onto M 10 tungsten particles First, right before use resuspend the particles in sterile water to a concentration of 100 milligrams per milliliter.
Then in a 0.6 milliliter fuge tube, add 25 microliters of particles, five microliters of DNA at one microgram per microliter, 25 microliters of 2.5 molar calcium chloride and 10 microliters of 100 millimolar sperm aine. Incubate the DNA preparation on ice for five minutes, then remove and discard 50 microliters of the solution on top of the particles. Re suspend the DNA coated particles which remain at the bottom of the tube by vortexing.
And immediately after vortexing, remove a two microliter Eloqua. Repeat this vortexing step. Every time an Eloqua is removed, the coated particles should be kept on ice and used within 15 minutes.
Now that the DNA is coated onto the particles, place two microliters of particles through the top of a syringe filter in the middle of a filter screen. Then place the syringe filter in the filter holding unit inside the particle inflow gun or PIG chamber. Place a lima bean co leadin a axial side up on a baffle which consists of a screen melted to the bottom of a beaker.
Place the baffle, which is used as a platform to support tissues during bombardment in the particle gun chamber. To bombard the tissue first open the valve leading to the vacuum to evacuate the chamber. When the vacuum reaches 760 millimeters of mercury, activate the solenoid and release the helium to propel the particles.
The helium pressure used to accelerate particles is 50 to 60 PSI. After the particle bombardment, close the vacuum line valve and release the vacuum using the exhaust valve. When the vacuum is released, open the chamber door to retrieve the bombarded co leadin and return the co leadin a dal side.
Up to OMS culture medium for quantification and expression profiling. Bombard a minimum of three co leadin for every DNA construct. A positive control, which gives a well-studied gene expression profile is also included.
The co leadin are now ready for imaging. Turn on the mercury lamp and wait for 30 minutes for the lamp to warm up. Turn on the power sources for the SPOT R-T-C-C-D camera and the motor controller, which drives the movement of the custom designed robotics platform, which holds eight plates.
Set the filter set for GFP detection by selecting the GFP two filter set at 480 nanometers excitation and 510 nanometers emission on an MZ L three dissecting microscope. Next, 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 collection.
Place the plates containing bombarded cuan on the robotics platform and tighten the lateral set screw to fasten them in position. Now open the custom software application that controls both the robotics platform and image acquisition. Also open the software white light controller switch using the software, orient the platform to the home position.
This is done before entering positions. For every coddle leadin in the motor 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 CU LEADIN for image collection. Using the live mode feature, move the cuddle leaden to the appropriate viewing area For the first cuddle leaden, use the manual knobs on the dissecting microscope to set the focus and set the magnification to 1.6 x. 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 set positions for all the remaining coens on the plates. Using the moving distance buttons, use the three manual leveling screws located below each plate on the robotics platform to adjust the focus for the remaining coan on the same plate. After all the coordinates of all the coan have been entered, save the position schedule coordinates.
Enter the image collection parameters such as type of light, which is blue for GFP and exposure setting, as well as specify where to store the images as described in the accompanying written protocol. 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 hours.
Generating well-defined gene expression profiles. Start image acquisition by opening the position schedule tab and then clicking on the run schedule button. Isolate the image collection system from extraneous light at the end of the 100 hour image collection.
The high resolution images at about five megabytes each are ready for image analysis. Use image ready to assemble the 100 sequential images from each folder. Import all the sequential images as frames and resize them to 800 by 600 pixels to speed image processing.
Next, use marks on a new introduced layer, which is visible in all frames and using selected GFP spots in the image reference. Manually align each image when alignment is complete, delete the layer used as a reference and save the file as a single PSD file. Also, export all the frames as a single MOV file using the highest resolution.
The files are now ready to start GFP quantification. Use image J to open the MOV file and crop a 400 by 300 pixel area. Then separate the sequential images into red, blue, and green channels using the RGB split tool for red and green channels.
Subtract the background fluorescence using the subtract measured ROI for each slice plugin tool. Calculate the GFP expression by adjusting the threshold levels and using a plugin designed to measure the mean gray scale value per pixel and the total number of GFP expressing pixels. Copy output values in image J.Finally paste the output values for green and red channels into Microsoft Office.
Excel for further data manipulation to give you an idea of the results of image alignment. This first animation shows the inaccuracy of the repositioning of the robotics platform during the 100 hour experiment. Spot movement is an artifact resulting from the inability of the robotics platform to return to the exact same position at each time point after manual image alignment, the GFP expressing cells can be readily observed and the unique features of this tracking system are easily visualized.
As this animation repeats note, the GFP expressing cells that are circled, the cell that is circled in red shows a very rapid decline in expression at 18 to 20 hours post bombardment while the cell that is circled in white shows a delayed onset of GFP expression. Aside from the animations, this procedure allows rapid quantification of GFP expression over time in the lima bean target tissue. These representative results show the comparative quantification of promoter intensity using the CAMV 35 S promoter and four different soybean promoters.
We have just shown you how to introduce DNA into catalin of germinate. The Lima vein seeds place the catalin on the robot, collect images for image analysis and analyze those images. We routinely use this procedure in our lab for validating clone plant promoters.
One can generate a large amount of data from the introduction of different plant promoter. However, this procedure is complicated with many steps that can go wrong when doing this procedure is important to start with fresh line bean seed or at least use high quality seed that had been stored properly. It's also important to get the bombard cut lid and set up on the robot quickly and to use the thick pet dish lid to minimize condensation problems.
And last, please note that the GFP that we use is another version called SGFP, front Gen HIN Lab at Harvard University. This GFP is not very stable and may be better for tracking rapid changes in gene expression. And that's itra us for watching us.
And good luck with your experiments.
