This procedure tests in vivo protein protein interaction between two Arabidopsis transcription regulators, seus spelled SEU, in short and lu holog or LUH. By utilizing the biomolecular fluorescence complementation seus is fused to the end terminal fragment of a yellow fluorescent protein, or YFP and LUH is fused to the C terminal fragment of Y-F-P-D-N-A of the two plasmid constructs. Seuss, NYFP and L-U-H-C-Y-F-P are bombarded into onion epidermal cells to test for their interaction.
An interaction between Seuss and LUH brings about the association of the end terminal and C terminal fragments of YFP in nuclei leading to YFP fluorescence in nuclei. After incubation for 16 to 20 hours in the dark, the onion epidermal peels are observed under a fluorescence microscope in order to visualize fluorescence. Hi, I'm Courtney Hollander from the laboratory of Dr.Zhi Lu at the Department of Cell Biology and Molecular Genetics at the University of Maryland.
Today we'll show you a procedure for testing protein-protein interaction in onion cells using the BioRad Helios gene gun. We use this procedure in our lab for testing protein-protein interactions and protein Localization in plant cells. So let's get started.
To Begin This procedure, prepare cartridges for the Helios gene gun following the Joe Video Protocol by Woods and Zeto 2008 that demonstrates in detail the cartridge preparation procedure for each preparation of cartridges. 50 micrograms of total plasma DNA are mixed with 12.5 milligrams of gold particles and 0.5 milligrams per milliliter of PVP solution for BIFC. Combining the same cartridge preparation, the two plasma DNAs of the putative interacting proteins Seuss fused to the N terminal half of YFP, abbreviated seus, NYFP and LU Homolog fused to the C terminal half of YFP abbreviated L-U-H-C-Y-F-P.
The combined amount is 50 micrograms per prep, yielding up to 50 cartridges at one microgram of DNA per 0.25 milligrams of gold per cartridge. Each cartridge is for one shot. After cartridge preparation, test the cartridges to learn if the gold particles are efficiently propelled.
Wrap a square piece of paraform around a Petri dish. Then set the helium pressure to between 150 and 200 PSI and fire the newly made cartridges into the param square. Observe faint gold particles on the parfum.
Different amounts of gold particles may be propelled onto the parfum at different helium pressures. However, if there's no significant difference, choose the lower pressure for bombardment. This test also indicates the area covered by each shot proceed.
To prepare the onion tissues, peel off the outer skin from a large yellow onion using a clean and sharp razor blade. Cut a two by eight centimeter rectangular area, four to five layers deep. Take out the rectangular onion tissue and discard the outer two layers.
Cut the remaining layers of the onion into about 1.5 centimeter square pieces. Place the onion pieces in a Petri dish containing circles of three mm filter paper. Moistened with sterile water cover the Petri dish.
The onion pieces are ready for bombardment. First Load the cartridges into the white round cartridge holders load cartridges containing different plasmid constructs into different cartridge holders. Each cartridge holder can hold up to 12 cartridges for 12 shots.
Three different holders are loaded here. The first holder has cartridges containing 35 S-G-F-P-A strong and constitutive promoter from the cauliflower mosaic virus. This plasmid serves as a positive control.
The second holder is loaded with the experimental cartridges containing an equal amount of S-E-U-N-Y-F-P and L-U-H-C-Y-F-P. The third cartridge holder is loaded with cartridges containing an equal molar mix of the puck spine vector and LUH puck spice. This also serves as a negative control.
Insert the battery and the fresh barrel liner into the Helios gene gun. Make sure that both the barrel liner and the gun have their respective O-rings attached. Next, insert an empty cartridge holder into the gene gun with the mark number 12 on the cartridge holder facing the top of the gun.
Advance the cartridge holder once or twice to make sure it is inserted correctly. Then attach the gun to the helium tank and adjust the pressure to between 150 and 200 PS.I wear ear protection and fire the gun a few times by holding down the side button while pulling the trigger. Firing the gun with the empty cartridge holder, loaded it cleans out the gun chamber and ensures the pressure is stable.
Before shooting the DNA, shooting a piece of onion here can be a negative control for background fluorescence. To shoot the DNA coated gold particles, remove the empty cartridge holder and insert the first loaded cartridge holder containing the positive control. Advance the cartridge holder so that the cartridge intended for firing is positioned at the bottom of the cartridge holder.
Now place onion pieces in the middle of an empty Petri dish with the innermost layer of the onion facing up. Rest the barrel liner of the gene gun on the Petri dish and fire the gun. Advance to the next cartridge and shoot a different onion piece.
Repeat this step until four to five pieces of onion are shot. Each onion piece is shot once only after shooting. Transfer the bombarded onion piece to a Petri dish containing moist three mm filter paper to shoot the experimental cartridges.
Change to cartridge Holden. Number two, remember to change the barrel liner to prevent contamination. Shoot four to five onion pieces as just shown for the positive control.
Then change to cartridge Holden. Number three, containing the negative control DNA. And again, shoot four to five onion pieces.
Change the barrel liner to prevent contamination. After bombardment. Cover the Petri dishes containing the onion pieces with lids.
Wrap them with paraform to prevent drying. Incubate the onion pieces at room temperature in the dark for 16 to 48 hours. Once the bombardment is done, turn off the gas and release the pressure before detaching the gene gun from the helium tank, unscrew the barrel liner and remove the cartridge holder and the used cartridges.
Finally, clean the cartridge holders and barrel liners by submerging them in a beaker with soapy water and sonicate. For 20 minutes. Afterwards, rinse with water to remove all soap residues.
Soak in 70%ethanol for an hour and then dry on paper towels. Proceed to observe the onion pieces 16 to 20 hours after bombardment. After incubating the bombarded onion pieces for 16 to 20 hours at room temperature, use forceps with flat ends to slowly peel a single cell layer off the inner epidermis of the onion.
This is the layer that directly faces the gun during the bombardment. Place the peeled layer onto a drop of water on a slide cover with a cover slip. But be careful to minimize bubbles.
Observe GFP or YFP fluorescence using a fluorescence microscope such as the zes axio observer, point Z one shown here under the bright field of the microscope. First check for cytoplasmic streaming to ensure that the cells are alive. To do so.
Expose the slide to the bright field light for a few minutes to warm up the onion tissue and then look for moving plastics cytoplasmic Streaming is not always easy to see, so do not get discouraged if it is not clearly visible. For fluorescence detection, use appropriate excitation and detection filters for GFP or YFP. Also check the negative controls, which are the onion pieces shot with a non fluorescent plasmid.
Faint yellow signals may appear from wounded cells and debris and onions. Also, check the positive controls. The plasmid containing constitutively expressed fluorescent reporter.
GFP fluorescence is seen in some but not all cells. Air bubbles may also yield background fluorescence. Visible GFP signals from onion epidermal cells indicate that the cartridges, the onion tissues, and the gene gun firing Are properly prepared and executed.
Shown here is the strong fluorescence observed From the 35 SGFP positive plasmid. The strong fluorescence is seen in the entire cell, including both the nucleus and cytoplasm. The brightfield image allows one to see the corresponding outline of the cells and nuclei.
Not every cell is transformed. The YFP fluorescent signal is detected in the onion cell nucleus when the experimental combination Seuss, NYFP and L-U-H-C-Y-F-P is bombarded into onion cells indicating that Seuss NYFP and L-U-H-C-Y-F-P interact in vivo. However, this interaction mediated fluorescence is significantly weaker than the 35 SGFP mediated fluorescence in the positive control.
Additionally, the SEUS and LUH transcription factors are localized in the nuclei. Thus, the YFP fluorescence is only observed in the nucleus. Further, the number of fluorescent cells is significantly lower than that of the 35 SGFP positive control.
This is because B FFC only works when both partner plasmids are expressed in the same onion cells. No fluorescent signal is detected in onions, bombarded with cartridge holder number three containing a mix of puck spine, vector and L-U-H-C-Y-F-P As is expected of the negative control. We've just shown you how to bombard onion Cells to test for protein protein interactions using the BioRad Helios gene gun system.
When doing this procedure, it's important to remember to use proper positive and negative controls and to know that BIFC fluorescence may be weaker in signal and fewer in the number of fluorescent cells. So that's it. Thanks for watching and good luck with your experiments.
