This method can help answer key questions in the epigenetics field such as the mechanisms of gene regulation and cause and effect relationships between different chromatin regulatory elements. The main advantage of this technique is that for the first time it is possible to module a chromatin loci with physiologically relevant endogenous enzymes. Generally individuals new to this method will struggle because mouse ESL culture is inherently difficult for those who have not been trained in the technique.
Further demonstration of this method is critical as cell viral infection steps are difficult to learn. The researcher must time two separate lines of cell culture and it is necessary to vigilantly monitor safety during these steps. To begin the protocol, passage 18 million of 293T HEK cells per 50 centimeter plate.
Aspirate the old media and add 10 milliliters of one X phosphate buffered saline, or PBS. Aspirate the PBS and add three milliliters of 0.05%trypsin. Incubate the cells in trypsin for eight minutes at 37 degrees Celsius.
Rock and tap the cells halfway through the incubation. The following day, when the cells have reached 60 to 80%confluency, perform a polyethylenimine, or PEI, transfection. Next, gently mix the DNA, PEI, and transfection media in a 15 milliliter conical tube.
Incubate the sample at room temperature for 15 minutes. After incubation, add the solution dropwise to the 15 centimeter plate. Then, incubate the sample for 16 hours in a 37 degree Celsius incubator with 5%carbon dioxide.
The next day, aspirate the media and gently replace it with prewarmed, fresh 293 HEK media to the 15 centimeter plates. After the media exchange, incubate the cells in a 37 degree Celsius incubator with 5%carbon dioxide for 48 hours. Count the cells with a hemocytometer.
Passage previously prepared mESC into a 12 well plate format with 100, 000 cells per well one day prior to infection. For cells grown in a 10 centimeter format, aspirate the old media, add five milliliters of one X PBS, aspirate the PBS, and add one milliliter of 0.25%trypsin. Incubate the cells in trypsin for eight minutes at 37 degrees Celsius and rock and tap the cells halfway through the incubation.
48 hours after the media exchange, remove and transfer the supernatant of the 293T HEK cells to a 50 milliliter conical tube. Centrifuge the supernatant at 300 times G for five minutes to pellet the cell debris. Next, filter the supernatant through a 0.45 micron membrane.
Concentrate the virus via ultracentrifuge with an SW32 rotor and spin the sample at 72, 000 times G for two and a half hours at four degrees Celsius. While the virus concentrates under ultracentrifugation, treat the CIA mESC's in the 12 well plate with fresh embryonic stem or ES media containing five micrograms per milliliter of Polybrene. When the virus concentration has finished, carefully aspirate the supernatant and carefully suspend the virus pellet in 100 microliters of one X PBS to avoid excess bubbles.
Add the virus and one X PBS to a 1.5 milliliter Microfuge tube. Vortex the tube at the lowest setting to fully suspend the virus. Spin down the tube in a mini tabletop centrifuge for five to 10 seconds to remove bubbles.
Add 30 microliters of the virus to each well of a 12 well plate. Swirl the plate and then centrifuge the samples at 1000 times G for 20 minutes. Place the 12 well plate back in the 37 degree Celsius incubator overnight.
The following morning, exchange the media with fresh ES media and allow infection to take place for 48 hours. After 48 hours of infection, select for cells that integrated the viral plasmid by adding the appropriate antibiotic. Change the media daily.
Keep the selection media on the cells for 72 to 96 hours in a 37 degree Celsius incubator with 5%carbon dioxide. Continue with chemical epigenetic modifier, or CEM, preparation and treatment by suspending the powdered CEM and dimethyl sulphoxide, or DMSO, to a stock concentration of one millimolar and a working concentration of 100 micromolar. After the cells have undergone selection for 72 to 96 hours, split the mESC's into a 12 well format with 100, 000 cells per well.
Incubate the cells in a 37 degree Celsius incubator with 5%carbon dioxide for 24 hours. After incubation, prepare a media solution of 100 nanomolar CEM with ES media. Use the media to feed the CIA mESC with one milliliter well.
Keep a well of cells without any added CEM's to serve as a control. Next, change the media by aspirating the old media and adding one milliliter per well off freshly prepared CEM containing or CEM free ES media every 24 hours for the duration of the experiment. After 48 hours of CEM treatment, image the cells without CEM treatment in the cells treated with 100 nanomolar CEM using a fluorescence microscope.
Take representative images using phase or bright-field to record the mESC morphology at 10 to 40 times magnification. The cells should have formed around colonies with a few differentiated cells. Under the FITC fluorescence channel, image both cell conditions.
Next, isolate the control and CEM treated cells for flow cytometry by aspirating the media, washing the cells with one milliliter of one X PBS, aspirating the PBS and adding 0.25 milliliters of 0.25%trypsin with EDTA for eight to 10 minutes. Confirm that the cells are no longer bunched in large clumps through the microscope using 20 times magnification. If the cells do not appear to be in a single cell suspension, gently pipette them up and down with a P1000 pipette to fully trypsinize the cells.
Quench the trypsin with one milliliter of fresh media and resuspend the cells at high speed with a serological pipette until the cells are in a single cell suspension. Then, spin down the cells at 300 times G for five minutes at room temperature. Aspirate the supernatant, wash the cells with one X PBS, and centrifuge the cells.
Aspirate the PBS supernatant and resuspend the cell pellet in 200 microliters of FACS buffer. Perform flow cytometry on approximately more than 50, 000 cells with the suspended cells and run the samples at a sheath speed of around 5, 000 cells per second. Finally, export the data for further analysis.
Phase images of the CEM system at the Oct4 locus and CIA mESC's are imaged after 48 hours of treatment with 100 nanomolar of CEM23 or with untreated cells indicate specific GFP repression. Fluorescent images show a bright GFP expression in the control cells and a reduced GFP expression in mESC's treated with CEM23. Flow cytometry consistently revealed a greater than 30%decrease in GFP expression in the CIA mESC's treated with 100 nanomolar of CEM23 for 48 hours.
Chromatin immunoprecipitation, or CHIP, reveled that 48 hours of treatment with 100 nanomolar of CEM23 decreased the level of H3K27ac at the target locus when compared to control cells. While attempting this procedure, it's important to remember to be careful with the mouse embryonic stem cells. If they differentiate during the protocol, the results will be nullified.
After its development, this technique paved the way for researchers in the field of synthetic biology to explore the in vivo mechanisms in the mammalian genome.
