This protocol allows to observe and detect transcription events and gives insights about the effects of DNA double-strand breaks on the ongoing transcription of a gene. The main advantage of the technique is the observation of individually-labeled RNA transcripts in single cells at time intervals of seconds over a total period of one hour or more. This method provides insight into the DNA damage response to investigate the crosstalk between transcription and cellular processes, such as DNA replication and DNA lesions.
Our advice is to observe cells using doxycycline and to perform calibration measurements to train the detection of the transcription site and labeled RNA molecules. In a 1.5 milliliter microcentrifuge tube, prepare solution A containing 150 microliters of reduced serum MEM plasma DNA and 2.5 micrograms per microliter of DNA in transfection helper reagent. In parallel, prepare solution B containing 150 microliters of reduced serum MEM and 1.5 micrograms per microliter of DNA in a lipid-based transfection reagent.
Incubate both solutions at room temperature for five minutes, then gently add solution A to solution B and incubate for 20 minutes at room temperature. To transfect the cells, add 300 microliters of mixed solution A and B dropwise to each dish and gently distribute it. Store the glass bottom dish inside a 100 millimeter standard cell culture dish and incubate it at 37 degrees Celsius in a humidified atmosphere with 5%carbon dioxide.
Prepare 1.5 milliliter microcentrifuge tube with 200 microliters of DMEM with HEPES without phenol red and supplemented with 10%charcoal-stripped fetal bovine serum, then add TA.Approximately one hour before starting microscopy observation, induce transcription of the reporter genes by adding doxycycline to the growth medium and gently mix by pipetting up and down with a 200 microliter micropipette. Transport the cells to the microscope at least 30 minutes before starting the observation and place the 100 millimeter dish with the cells inside the preheated large microscope incubation chamber. Place the microcentrifuge tube with the pre-diluted TA inside the large microscope environmental chamber to warm it to 37 degrees Celsius.
Replace the lid of the glass bottom dish with a lid that has a drilled three millimeter diameter hole. Select the 100X oil immersion objective in the microscope control panel and apply a drop of immersion oil to the objective. Set the glass bottom dish with the cells inside the microscope stage incubation chamber and lock it in place, then close the lid of the stage incubator and all doors of the microscope housing.
Start the microscope operating and control software, open the focus control window and click on the scope pane. In the emission selection pane, click the 100%eye box to set the ocular beam path for direct sample observation by eye. In the filter set menu, switch to eye filter set and click Brightfield, then press the open Brightfield button.
Move the microscope objective towards the glass bottom dish until the oil touches the glass. Look through the oculars and manually focus on the plane of the cells, then switch off the open Brightfield button. Leave the cells for 30 minutes before starting the experimental observations to allow them to adapt to the environmental conditions and prevent focal drift during imaging due to temperature gradients.
Set up a 200 microliter micropipette and 200 microliter filter tips at room temperature. In the focus control window of the microscope control software, set the laser intensity to 5%and enter a value of 50 milliseconds for the exposure time. Open the capture window to adjust the settings to perform an automated image acquisition of three-dimensional timelapses.
Select the 3D capture acquisition type and set 12 to 16 optical slices separated by 0.4 micrometers. Tick the checkboxes of range around current and return to current position after capture. In the timelapse capture pane, enter a value of 120 for the number of time points and 30 seconds for the interval.
Select the confocal filter set according to the transected fluorescent protein labels as mentioned in the text manuscript and set the exposure time for each channel to 50 milliseconds. Use the setting current for the laser power to use the value of 5%selected in the focus window. In the focus control window, go to the camera pane, select the scale image display control and choose the manual button to set up a fixed range of image intensities to be displayed.
Select the cells for the 3D timelapse imaging of transcription sites upon induction of a DNA double-strand break. Screen the cells and select three fields of view according to the conditions described in the discussion of the text. Focus each selected cell previously located in the center of the field of view with the transcription site in the middle plane of the Z-stack.
Mark each XYZ position in the XY plane of the focus control window by clicking set point. Add 200 microliters of the pre-diluted TA to the cells and start the 3D time series imaging by clicking on start on the capture window. Save the imaging data in the microscope control software data format on the microscope control computer hard drive.
Add 0.5 micrograms per milliliter of doxycycline to the growth medium of the cells one hour before starting the microscopy image acquisition. Mount the glass bottom dish inside the microscope stage incubation chamber and prepare the image acquisition as demonstrated previously. Use the same laser intensity and exposure settings as before.
Set the capture settings for 2D time series and set 120 time points at intervals of 500 milliseconds in the timelapse capture panel. Acquire dozens of calibration time series from multiple positions in order to generate datasets to count several hundred single transcript TFI measurements. Using this protocol, a graph is obtained displaying the number of fluorescently-labeled reporter gene transcripts over time with a temporal resolution of seconds over periods of up to hours.
The time course of TFI values of a PROM reporter gene transcription site labeled by the accumulation of MS2 coat protein labeled with the green fluorescent protein molecules on nascent transcripts is shown here. The induction of a single DSB in the reporter genes makes it possible to study its impact on the ongoing reporter gene transcription and the monitoring of transcription events emerging from the DSB site, namely break-induced transcription. TA addition and induction of a DSB leads to a suppression of PROM reporter gene transcription after around 11 minutes that is not restored until 60 minutes.
The complete recovery of the PP7-RFP signal shows break-induced transcription initiation. The EXON 2 antisense reporter gene shows the promoter-driven transcriptions termination, which is then replaced by antisense break-induced transcription as revealed by the accumulation of MS2 coat protein labeled with a red fluorescent protein binding to RNA generated from antisense MS2 stem loop sequences. Selection of the cells for imaging, set up the treated timelapse imaging adjusting the Z position of the labeled transcription site on each XY position into the center of the Z stack to be acquired.
And finally, the addition of the TA diluted in growth medium must be executed with extreme care to prevent any shifts of the pre-selected positions with cells to be imaged. The imaging of single RNA transcripts in 3D over time in live cells can be applied to study RNA transcription during DNA replication or changes of transcription during the cell cycle progression.
