The overall goal of this in vitro bioluminescence assay is to determine the effects of different environmental circadian disruptors and enhancers on the cellular circadian rhythm of mammary epithelial cells from mammary glands at normal or diseased states. This method can help answer key questions in the environmental health field such as whether environmental factors including chemical compounds affect the cellular circadian rhythm. So, the main advantage of this assay is that it can be readily adapted to a variety of cell types so we can screen for environmental modulators of circadian clocks in different organs.

Demonstrating the procedure with me is Brian Estrella, a graduate student in our lab. The human PER2 promoter fragment is obtained from a customized vector that contains human PER2P at the site between SecI and HindIII in the multiple cloning region. To cut the human PER2P fragment out from the vector, mix the following in a DNA/RNA and nucleotide-free microcentrifuge tube, 2.5 microliters of restriction enzyme buffer, 10 microliters of vector, one microliter of SacI restriction enzyme, one microliter of HindIII restriction enzyme, and ultrapure water to 25 microliters.

Incubate in a heating block at 37 degrees Celsius for 90 minutes. When the restriction digest is complete, run the whole volume of the reaction on a 0.7%agarose gel containing 0001%ethidium bromide to separate human PER2P from the vector. Cut a piece of agarose gel containing a fluorescence band at 941 base pairs, purify the human PER2P fragments from the gel, and quantify the DNA as described in the text protocol.

The next step is to clone human PER2P into a vector with destabilized fire flow luciferase. Linearize one microgram of the vector by restriction digest with SacI and HindIII as shown previously. Run the whole volume of the reaction on a gel containing ethidium bromide and then purify and quantify the vector DNA as before.

To ligate human PER2P into the linearized vector, gently mix the following in a microcentrifuge tube, two microliters of T4 DNA ligase reaction buffer, 3.26 microliters of human PER2P, one microliter of linearized vector, and water to 20 microliters. Then add one microliter of T4 DNA ligase, mix gently, and incubate at 16 degrees Celsius in a thermal cycler overnight. On the following day, chill the ligated vector on ice.

Prior to the transformation, set a water bath at 42 degrees Celsius, warm up SOC medium at room temperature, and warm up LB agar plates in the 37 degree Celsius incubator. Thaw two vials of competent E.coli cells on ice. To start the transformation, add six microliters of ligated vector to one tube of chemically competent E.coli and gently flip the tube to mix.

As a negative control, add one microliter of empty vector to another tube of chemically competent E.coli and incubate the tubes on ice for 30 minutes. After 30 minutes, heat shock in the 42 degrees Celsius water bath for 30 seconds without shaking and then return the tubes to ice. Add 250 microliters of SOC medium to each tube and incubate at 37 degrees Celsius with shaking at 200 rpm for one hour.

Next, evenly spread 10 to 50 microliters of each transformed E.coli reaction on the surface of an LB agar plate. Put the plates upside down in a 37 degree Celsius incubator overnight. On the following day, use a sterilized inoculating loop to pick three to six white colonies from each plate and release each colony into a culture tube containing four milliliters of LB culture medium with 50 micrograms per milliliter of ampicillin.

Incubate the tubes at 37 degrees Celsius with shaking at 200 rpm overnight. Use two milliliters of each four milliliters of cultured E.coli for DNA extraction. After verifying the insert as described in the text protocol, amplify the selected E.coli by adding the leftover two milliliters of cultured E.coli into 200 milliliters of LB culture medium containing 50 micrograms per milliliter of ampicillin and incubating at 37 degrees Celsius with shaking at 200 rpm overnight.

Plasma DNA is then extracted with an endotoxin-free plasmid Maxiprep kit per the manufacturer's instruction. After quantifying the plasma DNA, aliquot and store at minus 80 degrees Celsius for future use. The mammary epithelial cells to be transfected with the constructed circadian vector are cultured with mammary epithelial cell growth medium containing mammary epithelial basal medium, growth supplements, and cholera toxin.

To dissociate the cells, incubate with 10 milliliters of Trypsin-EDTA for about 20 minutes and then neutralize the Trypsin by adding 10 milliliters of Trypsin neutralizing solution. Transfer the cells to a 50 milliliter centrifuge tube. Rinse the dish with 10 milliliters of buffered saline solution and add the rinse to the 50 milliliter tube.

Spin down the cells at 1, 500 rpm for 10 minutes. Remove the supernatant and add three milliliters of MEGM to resuspend the cells. Count the cells with a hemacytometer under an inverted microscope.

After calculating the concentration of the single cell suspension, seed two times 10 to the fifth cells evenly per 35 millimeter culture dish with MEGM. Swirl the plates thoroughly to obtain an even distribution of cells in each plate. Grow the cells in the cell culture incubator to 20 to 30%confluence.

On the following day, warm up reduced serum medium in a 37 degree Celsius water bath and the transfection reagent at room temperature for 30 minutes. Bring the plasma DNA to room temperature. Dilute the plasma DNA to 30 nanograms per microliter with endotoxin-free Tris-EDTA buffer and keep at room temperature.

Prepare each plasmid transfection mixture in a 1.5 milliliter microcentrifuge tube. First, add 63.6 microliters of warm reduced serum media. Then, add 33.4%microliters of plasma DNA and mix gently by pipetting.

Lastly, add three microliters of transfection reagent directly into the center of the tube without touching the wall. Mix gently by pipetting and keep at room temperature for 30 minutes. Drop the entire plasma transfection mixture directly onto the center surface of the medium in the plate and mix gently by shaking the dish.

Culture the cells in a carbon dioxide incubator for an additional 48 to 72 hours. 48 to 72 hours after transfection when the cultured cells are about 70%confluent, replace the medium with MEBM without growth factors and culture the cells for an additional 24 hours. After 24 hours of starvation, add a synchronization agent in MEBM to the cells.

50%horse serum is used as the synchronization agent in this experiment. Incubate for 1-1/2 hours in a carbon dioxide incubator. Prepare recording medium.

When the treatment with the synchronization agent is complete, wash the cells three times with warm D-PBS. Then add two milliliters of freshly prepared recording medium containing luciferin. Seal the dish with a sterilized cover glass using silicone grease to prevent evaporation.

Label the dish on the side. Place the sealed dish in the seat inside the luminometer and perform data collection and analysis as described in the text protocol. In this in vitro model, untreated transiently transfected cells showed at least two complete cycles of luminescence signaling after synchronization.

Cells exposed to the mutagen NMU showed disrupted circadian rhythm at 0.5 millimolar concentration as reflected by the loss of luminescence signaling after synchronization. Inhibition of Cert1 activity with X257 or Cambinol similarly disrupted circadian rhythm by dampening the subsequent circadian cycle. Importantly, the addition of chemopreventive MSC to the culture medium restored the rhythm disrupted by 0.5 millimolar NMU and prevented the disruptive effects of the Cert1 inhibitors.

In stably transfected cells, both NMU and Cert1 inhibitors disrupted circadian rhythm. MSC restored circadian rhythms in cells pretreated with NMU, X275, or Cambinol. The bioluminescence intensity was higher but the rhythm was sustained for a shorter time in transiently transfected versus stably transfected cells, but the overall results are consistent.

The cellular circadian rhythm can be quantified in circadian parameters such as period and phase using LumiCycle analysis software. Once mastered, this technique is simple, fast, and safe. While attempting this procedure, it's important to track if the cells are proliferating since the transfection efficiency is very low in nondividing cells such as neuronal cells in which case a virus-spaced transfection method could be considered.

After transfection, other methods like treatment with different chemicals or genetic modification can be performed to answer additional questions like how is the cellular circadian rhythm affected by environmental and genetic factors. This assay can be used to investigate the impact of disrupted circadian rhythm on cellular function. By studying the underlying mechanisms, we can develop better intervention strategies for those at increased risk of breast cancer due to abnormal work schedules.

After watching this video, you should have a good understanding of how to establish and validate the in vitro bioluminescence assay to determine the circadian rhythm in the cells of interest. Don't forget that working with a UV illuminator can be extremely hazardous and precautions such as using a UV protective face shield should always be taken while performing this procedure. Thanks for watching and good luck with your experiments.