Begin this procedure with preparation of mouse tendon fibroblasts, as described in the text protocol. Harvest the cells from a 10-centimeter plate by first aspirating the media. Then add one milliliter of 0.05%trypsin EDTA and incubate for two to five minutes at 37-degrees Celsius.
Following incubation, quench the trypsin with nine milliliters of DMEM-10, and collect the resuspended cells in a 15-milliliter conical tube. Dilute the cells with DMEM-10 to a final concentration of one cell per 100 microliters. Then transfer the cells to 96 well plates by pipetting 100 microliters of the suspension into each well.
Incubate the cells at 37 degrees Celsius until the wells are confluent, which can take up to two to three weeks. Replace the media with fresh DMEM-10 every three to five days. When confluent, mobilize the cells with 20 microliters 0.05%trypsin EDTA per well, and split them into two duplicate sets of 96 well plates.
Assay for luciferase activity in one set of plates. Use a homogenous Firefly luciferase assay that does not require media removal, following the protocol included with the luciferase assay kit. Use the assay results to select clones with no detectable luciferase activity from the remaining set of plates.
Expand these clones first to a 24 well plate, and then to a six well plate. Perform the same for a clone with the highest luciferase activity. This will be used as a positive control for validation purposes.
Following expansion in the six well plate, harvest an aliquot of each well for a Western blot. Mobilize the cells with 200 microliters of 0.05%trypsin EDTA. Incubate the cells for two to five minutes at 37 degrees Celsius.
Quench the cells with two milliliters of DMEM-10, and transfer half of the cells to another six well plate. Wait until the cells attach to the bottom of the plate. Then wash the cells once with PBS before the lysing for the Western blot.
Plate several negative clones at 0.2 million cells per well in a six well plate. Treat the cells once with 10 micromolar 5-azacytidine. Then incubate for 72 hours without changing the media before assaying for luciferase activity, as before.
Next, choose one negative clone that gained luciferase activity with 5-azacytidine for the screen, and begin expanding it to multiple 10-centimeter plates. Freeze the remaining negative clones in liquid nitrogen as a backup. The positive clone can be maintained in the tissue culture or frozen until further use.
In the morning, plate one million inactive X cells onto 10-centimeter tissue culture plates. In the afternoon, infect the plates by replacing the media with fresh DMEM-10 containing a predetermined amount of viral supernatant and five to 10 micrograms per milliliter of polybrene. After 18 to 24 hours, aspirate the media and add 10 milliliters of fresh DMEM-10 to each plate.
Culture plates for another 48 hours. In the morning, mobilize the cells using one milliliter of 0.05%trypsin EDTA per plate, and quench the cells with 9 milliliters of DMEM-10 per plate. Run the suspension through a 100-micron mesh filter.
Pull the filtered suspensions together, and seed two sets of 10-centimeter plates at one million cells per plate. In the afternoon, replace the media in one set of plates with DMEM-10 containing 15 micrograms per milliliter of Hygromycin B.In the other set, replace the media with fresh DMEM-10 only. After 48 hours, mobilize the cells from Hygromycin B untreated plates using one milliliter of 0.05%trypsin EDTA.
Incubate for two to five minutes at 37 degrees Celsius. Next, quench the cells with 9 milliliters of DMEM-10. Pull the individual samples, and proceed immediately with extracting DNA.
Continue culturing the Hygromycin B selection set for six more days, replenishing the selection media with fresh DMEM-10 containing Hygromycin B every other day. On day six, replace the selection media with fresh DMEM-10 only. Allow four days for recovery, and then harvest the post-selection sample DNA as before.
Proceed to identify hairpins enriched in the post-selection samples, as described in the text protocol. To validate the hits, first seed eight million 293-FT cells per plate on 10-centimeter plates. The following day, replace the media with nine milliliters of antibiotic-free DMEM-10 per plate 30 minutes before transection.
Next, add 0.5 milliliters of transfection mix one to each aliquot of transfection mix two, and gently mix by pipetting. Then incubate the mixture at room temperature for 20 minutes. Apply the mixture onto each plate in a drop-wise fashion, and swirl the plate gently to mix.
The following day, replace the media with five milliliters of fresh DMEM-10.48 hours after initial transfection, collect the lentiviral supernatant, and run it through a 0.22-micron filter that has been prewet with DMEM-10 to maximize recovery. The next morning, plate 0.1 million inactive X cells per well onto six well plates. In the afternoon, infect the plates by replacing the media with two milliliters of fresh DMEM-10 containing lentiviral supernatant and five to 10 micrograms per milliliter of polybrene.
Infect a control well with viral supernatant produced from an empty lentiviral vector. The following day, replace the media with two milliliters of DMEM-10 containing one milligram per milliliter of puromycin. Use selecting for four days.
After four days, replace the media with two milliliters of fresh DMEM-10, and allow 48 to 72 hours for recovery. After mobilizing the cells with 150 microliters of 0.05%trypsin EDTA, incubate them for two to five minutes at 37 degrees Celsius. Quench the cells with 1.5 milliliters of DMEM-10 before transferring to conical tubes and centrifuging the cells at room temperature at 500 times G for 10 minutes.
Remove the media and resuspended the cells in 100 microliters of lysis buffer provided in the luciferase assay kit. Incubate the suspension at room temperature for five minutes. After five minutes, transfer the suspension to a white 96 well plate.
Perform the assay in the darkroom, following the protocol included in the kit. Use active X cells as a positive control and negative X cells infected with an empty lentiviral vector as a negative control. Immortalized mouse tendon fibroblast clones show either positive or negative Firefly luciferase activity, consistent with the placement of the reporter on the active or inactive X chromosome, respectively.
MeCP2 expression, as determined by the Western blot, exhibits the reciprocal pattern. Note that Firefly luciferase activity can be induced in negative clones with administration of 5-azacitidine. If present on the active X chromosome, the Hygromycin resistance gene confers a survival advantage under Hygromycin selection, when compared to the cells with the gene on the inactive X chromosome.
After watching this video, you should have a good understanding of how to design and perform a screen for regulators of X-chromosome inactivation using reporters selectively inserted on inactive X chromosome. My laboratory has previously carried out reported base screens for chromatin regulators in yeast, and our success with this approach inspired me to design this screen for regulators of X-chromosome inactivation in mammalian cells. Following this procedure, other methods, such as using small, interfering RNAs, CRISPR, small molecules can be performed to help answer additional questions regarding X-chromosome inactivation in mammalian cells.
