The overall goal of this series of experiments is to introduce an engineered split-TET2 enzyme called CiDER into mammalian cells for inducible DNA modification such as hydroxymethylation as well as epigenetic remodeling. This method can help answer key questions in the epigenetics field such as The main advantage of this technique is that the engineered split-TET2 enzyme allow temporal control of 5-methylcytosine oxidation and subsequent remodeling of epigenetic states in mammalian cells with chemical additives. To begin this procedure, culture an adherent cell line in DMEM supplemented with 10%heat-inactivated FBS as well as 100 units per milliliter of penicillin and streptomycin.
Incubate the cells at 37 degrees Celsius with 5%CO2. Transfect the cells with the CiDER plasmid as described in the text protocol. Then incubate the cells overnight at 37 degrees Celsius.
The following day, replace the transfection reagent-containing media with fresh complete DMEM. To chemically induce the cells, add 20 microliters of diluted Rapamycin to the transfected cells maintained in two milliliters of medium to reach a final concentration of 200 nanomolar. To perform flow cytometry, resuspend the transfected and Rapamycin-induced cells in FACS buffer.
For the control group, use CiDER-expressing cells without Rapamycin treatment. Fix and permeabilize the cells as described in the text protocol. Then add two normal hydrochloric acid to the cells to denature the DNA for 10 minutes.
After neutralizing and blocking the cells as described in the text protocol, add the diluted anti-5hmC antibody to the cells. Incubate the cells for one hour at room temperature or overnight at four degrees Celsius. Following incubation, wash the cells with FACS buffer three times.
Remove the FACS buffer thoroughly. Add a diluted fluorophore conjugated secondary antibody for FACS analysis and incubate for one hour at room temperature. Then wash the cells with FACS buffer three times.
Following the wash, the samples are ready for FACS analysis. Isolate genomic DNA from transfected and Rapamycin-induced cells using a commercial DNA extraction kit. For the control group, use CiDER transfected cells without Rapamycin treatment.
Heat the vacuum oven to 80 degrees Celsius. Pre-soak the nitrocellulose membrane in double distilled water and then in 6X SSC buffer for 10 minutes. Next, load 60 microliters of equal amounts of DNA to a 96-well PCR plate.
Add 30 microliters of TE buffer to the rest wells. Make two-fold serial dilutions vertically on the 96-well plate by transferring 30 microliters of solution in row one to the same column position in row two. Mix again and transfer 30 microliters of solution to the wells in the subsequent row until the boundary is reached.
Then remove the last 30 microliters from the well. Next, add 20 microliters of one molar sodium hydroxide and 10 millimolar EDTA to each well. Seal the plate and heat the mixture for 10 minutes at 95 degrees Celsius to completely denature the DNA duplex.
Immediately cool down the samples on ice. Add 50 microliters of ice cold two molar ammonium acetate to each well and incubate on ice for 10 minutes. Meanwhile, assemble the dot-blot apparatus with the pre-soaked membrane.
Remove the residual buffer using a full vacuum. Wash the membrane by adding 200 microliters of TE buffer to each well of the dot-blot apparatus. Turn on the vacuum to remove TE buffer.
Next, load the denatured DNA to each well of the dot-blot apparatus. Turn on the vacuum to remove solution. Wash the membrane by adding 200 microliters of 2X SSC and remove residual solutions completely using a vacuum.
Disassemble the dot-blot apparatus. Then take out the membrane and rinse the whole membrane with 20 milliliters of 2X SSC. Air dry the membrane for 20 minutes.
To further dry the membrane, bake it at 80 degrees under vacuum for two hours. Add the blocking solution to the membrane and incubate for one hour at room temperature. After discarding the blocking solution, add the diluted 5-hmC or 5-mC antibodies to the membrane and incubate overnight at four degrees Celsius.
Wash the membrane with TBST three times for 10 minutes to remove residual antibodies. After removing the TBST thoroughly, add an HRP conjugated secondary antibody to the membrane. Incubate the membrane for one hour at room temperature.
Wash the membrane with TBST three times for 10 minutes. Finally, add ECL western blotting substrate to the membrane for visualization of 5-hmC signals using a chemiluminescence detector. A representative quantification result of CiDER-mediated 5-hmC production by flow cytometry is shown here.
Only cells expressing CiDER under Rapamycin-treated conditions show significant increase of 5-hmC levels. A representative result of the global change of 5-hmC level in the whole cell population by dot-blot assay is shown here. The loading control is visualized by ethylene blue staining of total amounts of input DNA.
The results demonstrate that Rapamycin treatment induces robust production of 5-hmC in CiDER-expressing HEK293T cells with negligible background activity. Once mastered, this technique can be done in a week if it is performed properly. After its development, this technique paved the way for researchers in the field of epigenetics to explore cellular function without altering the genetic code and to probe the epigenotype and phenotype relations in various biological systems.
