This protocol details the generation of luciferase reporter cell lines and the subsequent monitoring of cell autonomous circadian rhythms of bioluminescence expression. First construct lentiviral luciferase expression reporters and produce lentiviral particles proceed to infect the cells of interest and amplify selected cultures. Then monitor the bioluminescence expression in synchronized reporter cells using an appropriate recording device.
Ultimately, bioluminescence recording is used to demonstrate cell autonomous circadian rhythms. The main advantage of this technique over existing methods like transient transfection and the germline protection is that VIR is mediated. Gene provides stable integration into cells genome, as well as greater efficiency and versatility.
This method Can help answer key questions in the field of cied clock research. For example, are there tissue specific circadian clocks? If so, what are the properties of tissue specific circadian clocks?
Though this method can provide insights into clock mechanisms. It can also be applied to other systems such as dynamics of the cell cycle, tumorgenesis, et cetera. We first had the idea for this method when we decided to study tissue or cell type specific securing clocks.
Demonstrating the procedure will be Dr.Chita Norm Napkin, a postdoc fellow, and Sandra, a graduate student from my lab Atory. Typically, a mammalian circadian reporter construct contains an expression cassette in which a circadian promoter is fused with the luciferase gene. In this lentiviral reporter, the destabilized luciferase is under the control of the mouse per two promoter.
For detailed instructions on constructing reporter vectors by recombination reaction, consult the accompanying manuscript for efficient transfection. Start with a culture of human embryonic kidney cells of low passage number that are 90 to 100%confluent. Now to coat six, well culture plates.
For the transfection, add one milliliter of 0.001%poly L lycine to each, well incubate it room temperature for 20 minutes and rinse once with PBS. Next, harvest the cells using trypsin and seed the cells onto each well of the pre-coated plates. Swirl the plates to obtain an even distribution of cells.
Verify that the seeded cells have reached confluence of 80 to 90%in a 1.5 milliliter micro centrifuge tube. Prepare the plasmid transfection mix of a lentiviral reporter plasmid, DNA and the three packaging vectors as a control for both transfection and subsequent infection. Include an additional well for a lentiviral GFP expression vector that harbors the enhanced green fluorescent protein under the control of the CMV promoter.
Next, add 100 microliters of 0.25 molar calcium chloride to the plasmids and mix thoroughly. Then add 100 microliters of two XBBS solution and mix gently but thoroughly. Incubate the DNA mix at room temperature for 15 minutes.
While waiting, replace the medium from the 2 9 3 T cells with two milliliters fresh medium. Return the plate to the incubator for at least 10 minutes to equilibrate medium pH. Next in a dropwise fashion, add the transfection mix to the cells, swirl the plate gently observe particle formation under a microscope, and then place the cells in an incubator overnight.
About 16 hours post transfection replenish the medium with two milliliters of fresh DMEM and culture overnight. Next, to assess transfection efficiency, evaluate the EGFP expression in the transfection control cells. Transfection efficiency of 90 to 100%with high EGFP expression is a reliable predictor of a good viral prep.
Harvest the medium containing secreted infectious viral particles centrifuge to remove the residual 2 9 3 T cells and collect the virus containing supernat. On day three seed approximately 12, 003 T three cells on a 12 well plate culture overnight to 20 to 30%co fluency and observe the cells on day four. Still on day four, add poly brain to the collected medium containing viral particles and mix well by pipetting.
Now aspirate the medium from the three T three cells and add one milliliter of viral mixture per well. Incubated 37 degrees Celsius overnight after washing cells once with PBS, replenish the medium and incubate the cells at 37 degrees Celsius overnight for recovery and growth. When confluent split the cells and culture overnight the following day.
To start selection of infected cells, add fresh medium containing 10 micrograms per milliliter. Blastic aside, The blast acid in kill car needs to be empirically determined for each cell line. Replenish the medium containing blastin every two to three days for continuous selection of antibiotic resistant cells.
Expressing clock reporters. After propagating the blastin resistant reporter cells culture into 35 millimeter culture dishes until confluent, We usually prepare three or more dishes for each reporter cell line under each condition for sarcy phenotype. After one wash with PBS add DMEM containing 10 micromolar forline or 200 nanomolar dexamethasone incubated 37 degrees Celsius for one hour to synchronize the cells.
Now place the cells in freshly made recording medium. Cover the culture dishes with 40 millimeter sterile cover slips and seal in place with vacuum grease to prevent evaporation. Load the dishes on the lumous cycle luminometer and start the realtime bioluminescence.
Recording For recording 96 old plate. With the GSR two recording device see accomp manuscript. We easily record the rhythm for five to seven days In the lum cycle analysis program.
First baseline fit the raw data. Then use the baseline subtracted data to fit to a sine wave and determine the required parameters For samples that show persistent rhythms. A goodness of feet up data than 90%is easily as achieved due to high transient Luminate.
Sense up and medium change. We usually exclude the first cycle of data from our analysis For the data presentation. Plot raw data against time when necessary.
Plot the baseline subtracted data to compare amplitude and phase in both transient transfection of 2 9 3 T cells and in infection of cell lines. Fluorescent images of the transfected and transduced GFP expressing cells indicate that the lentiviral mediated gene delivery system is highly efficient. The core feedback loop of a circadian clock consists of transcriptional factors that act on the circadian enhancer elements to produce rhythmic gene expression.
Here, four different reporter constructs in three T three cells produce distinct phases of reporter expression. Novel intermediate phases can be generated with combinatorial regulation by multiple circadian elements using gene knockdown via RNAi and pharmacologically active compounds. This experiment used the lum cycle luminometer for bioluminescence recording of cells in 35 millimeter dishes in U2 OS cells.
Irna was used to illuminate the knockdown effects of cry one and cry two genes. Here a synergy luminometer was used for bioluminescence recording of cells. In a 96 well plate in three T three reporter cells.
RNAs were used to evaluate the effects of triune knockdown on cellular rhythms. In this example, the UX system and a tecan luminometer were used for bioluminescence recording of cells in 3 84. While plates as indicated selected, small molecules can pharmacologically target and perturb protein function in cellular rhythms of U2 OS reporter cells.
After its development. This technique paved the way for research in the field of circadian biology to explore clock mechanisms and physiology in different cell and tissue types. After Watching this video, it should have a good understanding of how to generate Lucifer, report cell lines and monitor the volumes.
And Don't forget that working with the VIR can be extremely hazard orders, and the precursion, such as personal protections and viral decontamination should always be taken while performing this procedure.
