Dec 04, 2019
Spectral Recording of Gene Expression History by Fluorescent Timer Protein
- Anna R. Tröscher1,
- Barbara Werner1,
- Nadia Kaouane1,
- Wulf Haubensak1
- 1Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), Campus Vienna Biocenter 1, 1030 Vienna, Austria
- BioTechniques
External link: https://www.future-science.com/doi/10.2144/btn-2019-0050
Protocol Citation: Anna R. Tröscher, Barbara Werner, Nadia Kaouane, Wulf Haubensak 2019. Spectral Recording of Gene Expression History by Fluorescent Timer Protein. protocols.io https://dx.doi.org/10.17504/protocols.io.8jvhun6
Manuscript citation:
Anna R Tröscher, Barbara Werner, Nadia Kaouane & Wulf Haubensa. Spectral recording of gene expression history by fluorescent timer protein. BIOTECHNIQUES. VOL. 67, NO. 4 | 6 Sep 2019 | doi.org/10.2144/btn-2019-0050
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 21, 2019
Last Modified: December 04, 2019
Protocol Integer ID: 29013
Keywords: flow cytometry, fluorescent timer protein, gene expression, immediate early genes, live cell imaging, spatio-temporal resolution
Abstract
Monitoring spatio-temporal patterns of gene expression by fluorescent proteins requires longitudinal observation, which is often difficult to implement. Here, we fuse a fluorescent timer (FT) protein with an immediate early gene (IEG) promoter to track live gene expression in single cells. This results in a stimulus- and time-dependent spectral shift from blue to red for subsequent monitoring with fluorescence activated cell sorting (FACS) and live cell imaging. This spectral shift enables imputing the time point of activity post-hoc to dissociate early and late responders from a single snapshot in time. Thus, we provide a tool for tracking stimulus-driven IEG expression and demonstrate proof of concept exploiting promoter::FT fusions, adding new dimensions to experiments that require reconstructing spatio-temporal patterns of gene expression in cells, tissues or living organisms.
Attachments
Guidelines
We do not go into details regarding the plasmid cloning process, as we welcome everyone who is interested in using it, to contact us and we will provide it. Therefore, we will only describe the cell culture experiments.
Mathematical extrapolation of cellular activity
For the mathematical differentiation of induced and noninduced cells, z-scores of the fluorescent intensity for each color were calculated from a total of 20 cells. The mean and SD for both colors were calculated separately, including all cells and time points of interest. Z-scores were then calculated by subtracting the value of interest by the mean and dividing through the SD of the respective color. To determine the time point of cellular activity of individual cells during the live cell imaging, we established a mathematical model that allows extrapolating gene activity depending on the ratio of blue to red fluorescence. Thus, the change of the fluorescent signal of blue and red over time was calculated. To this end, the logarithm of the ratio of the fluorescent intensities of five randomly chosen cells was plotted over time. In a next step, the linear curve function was used to extrapolate the time point of cellular activity of five cells (again randomly chosen) from the same experiment with unknown induction time points. To verify this approach, we repeated these calculations with a larger dataset with a total of 18 randomly chosen cells from an independent experiment. For the mathematical differentiation of early and late responders, the median response rate to the stimulus was used as a cut-off. If the calculated response was before the median, cells were grouped as early responders, whereas cells, for which the calculated response time was more or equal than the median, were allocated to the late responder group.
Troubleshooting
If you see a positive signal in your - TPA cells, try handling your cells with even more care, as the FT expression can be activated very easily. Make sure medium is really on 37 °C and not too cool before you add it. Avoid any type of stress for your cells as good as possible.
Materials
MATERIALS
6-well cell culture plates Eppendorf
DMEM Life Technologies
FCS (Fetal Calf Serum) Life Technologies
Pen/Strep (Penicillin/Streptomycin)Life Technologies
L-Glutamine Life Technologies
TransIT®-LT1 Transfection ReagentMirus BioCatalog #MIR 2304
Opti-MEM I reduced serum medium Life Technologies
Phorbol-12-myristate 13-acetate Merck MilliporeSigma (Sigma-Aldrich)Catalog #P8139
PBSLife Technologies
Trypsin/EDTA Life Technologies
- The FT-ESARE/ArcMin plasmid can be obtained from the authors directly.
- HeLa cells (ATCC, CCL2)
Recipes
standard medium: DMEM + 10 Mass Percent FCS + 1 Mass Percent Pen/Strep + 1 Mass Percent L-Glutamine
Required Equipment
- Cell Culture working space (incubator, hood, centrifuge)
- FACS (BD LSR Fortessa2, BD Biosciences)
- Chambered cell culture slides (8-well Cell Culture Slides, CCS-8, MatTek Corporation)
- Spinning Disk microscope (Spinning Disk Confocal UltraView Vox, controlled by software Volocity)
Safety warnings
Please refer to the Safety Data Sheets associated with each reagent for safety information.
Cell Culture and Transfection
Cell Culture and Transfection
Grow HeLa cells overnight in standard incubator conditions (37.5 °C , 5 % CO2) on 6-well plate in standard medium (DMEM + 10 % FCS + 1 % Pen/Strep + 1 % L-Glutamine) to a confluency of about 80 %.
Transfection with the TransIT-LT1 transfection reagent according to manufacturer: To 1 µg/µl plasmid in Opti-MEM I reduced serum medium , add TransIT-LT1 reagent.
Incubate for 00:30:00 at Room temperature .
Add reagent to cells for 24:00:00 .
Remove medium containing transfection reagent, wash with PBS and add fresh standard medium.
There are alternative options for the following steps:
- Fluorescence-activated cell sorting (FACS) (alternative I)
- Life-cell imaging (LCI) (alternative II)
Fluorescence activated cell sorting13 steps
alternative I
FACS
FACS
Leave cells undisturbed until induction ( 48:00:00 are recommended).
Baseline measurement: cells dislodged with trypsin and fluorescence and cell number determined by FACS.
Induction with TPA.
Safety information
TPA is highly cancerogenic so wear respective protective gear!
Note
TPA is very volatile: make sure you only add TPA to the wells, in which you want to induce the cells!
Grow + and - TPA cells in separate 6-well plates to guarantee no TPA spills over into - TPA wells, as also the smallest amount of TPA induce IEG expression.
Incubate for 00:40:00 with 100 ng/ml TPA diluted in standard medium .
Carefully remove all TPA and also remove medium of - TPA cells.
Wash with PBS.
Add fresh medium or add about 2 mL trypsin until cells are detached from the plate.
Add 2 mL standard medium to the cells for deactivation.
Pellet cells by centrifugation.
Resuspend in PBS.
Perform FACS measurements at the time points of your choice.
Note
We recommend every two hours for the first 9h and again at 24h post-induction.
You can analyze FACS data with any standard FACS analysis program (e.g. FlowJo).