Team:MIT/Experiments/Promoters/Experiment-Details
Promoter Testing Workflow
Our synthetic mammalian promoters were designed and constructed by us the students using standard Golden Gate Assembly and LR Cloning starting from DNA ordered from IDT. After construction was complete, our promoters were tested in a variety of cell lines under different hormone conditions. Cells were first seeded into a 24 well plate and allowed to grow for one day in order to reach confluency. The next day cells were transiently transfected with a promoter readout plasmid that contained our promoter upstream of eYFP, as well as with a constitutively active transfection marker hEF1a mKate. Depending on the cell line and the ease with which it could be transfected, either cationic lipid transfection or electroporation were used to deliver plasmid DNA to cells.
Twelve to twenty four hours after transfection, cells were induced with either estradiol (E2) , an estrogen hormone produce by the ovaries, or medroxyprogesterone actetate (MPA) , a progesterone analog. Twelve to twenty four hours after hormone induction, cells were prepared for flow cytometry in order to probe for the response to hormone on a population scale. Flow cytometry data was then analyzed using the open source Python toolbox, Cytoflow, which enabled us to bin our data by the transfection marker hEF1a mKate. This kind of analysis proves critical for comparing populations of cells that have been transiently transfected, because transient transfection procedures result in an uneven distribution of plasmids across the cell population. Binning the data allows us to compare cells which received roughly the same copy numbers of our two plasmids across different populations. Else, comparing cells that received a greater copy number of the plasmid to those which received less could result in skewing of the data towards a positive result.
Since we plan on deploying our diagnostic in an endometrial biopsy which contains a heterogeneous population of epithelial and stromal cells, it was critical to test the functionality of our promoters in a variety of cell lines. The cell lines our promoters were deployed in include the following:
- MCF7. A standard steroid receptor positive cell line derived from breast adenocarcinoma that was used to prototype our estrogen and progesterone signaling sensors.
- Ishikawa. An epithelial cell line derived from endometrial adenocarcinoma. These cells were used to test how our promoters would function in epithelial tissue from an endometrial biopsy.
- tHESC. A cell line of TERT-immortalized Human Endometrial Stromal Cells. These cells were used to test how our promoters would function in stromal tissue from an endometrial biopsy.
The MCF7, Ishikawa, and tHESC cell lines were provided through a collaboration that we initiated with the Griffith Lab at MIT affiliated with the Center for Gynepathology Research at MIT.
Promoter Architectures
We tested several promoters that contained binding sites (ERE) for the human estrogen receptor and binding sites (PRE) for the human progesterone receptor upstream of a minimal CMV promoter:
- pERE-#n. A promoter that contains n binding sites for ER in front of a minimal CMV promoter. n = 3, 5, 6.
- pPRE-#n. A promoter that contains n binding sites for ER in front of a minimal CMV promoter. n = 3, 4.
- pHybrid. A promoter that contains 5 binding sites for ER and 5 binding sites for ER alternatively spaced. This design roughly preserves distance from the TATA box between binding sites in the hybrid promoter and those tested in other constructs.
Figure . Promoter architectures with different copy numbers of binding sites for ER and PR.
Sensing Estrogen Signaling in MCF-7
We first deployed our sensors for estrogen signaling in the standard, easy-to-transfect cell line MCF7. Given that this cell line is known to over-express estrogen receptor, MCF7 provided a convenient platform for demonstrating the functionality of our promoters where we expected to see a large response.
Testing On - Off Functionality of Estrogen Sensitive Promoters
In our first set of experiments in this cell line, we sought to demonstrate our promoters basic on - off functionality by comparing uninduced cells to cells induced with 5 nM of E2. pERE3 demonstrated a fold increase in activity between the induced and uninduced populations; pERE5 demonstrated fold increase in activity ; pERE6 demonstrated a fold increase in activity .
Figure . Each of the three panels represent results for promoters pERE3, pERE5, pERE6. The y-axis represents the measured yellow fluorescence intensity from the eYFP on our reporter plasmid, whereas the x-axis represents the measured red fluorescence intensity from the mKate on our constitutively active transfection marker. Since transient transfection results in an uneven distribution of plasmids, it is important to bin our data by transfection marker so that cells which received roughly the same number of plasmids can be compared against one another.
Finer Characterization Studies
In our next set of experiments, we sought to obtain a finer characterization of our promoters through exposing transfected cells to a sweep of E2 concentrations including 0.25 nM, 0.5 nM, 1 nM, 2.5 nM, 5 nM, 10 nM. We had hypothesized that our promoters would demonstrate a graded response in eYFP production to this graded induction of E2 levels.
Figure . Each of the three panels represent the results of fine induction experiments for promoters pERE3, pERE5, pERE6. Colored contours represent different levels of E2 induction ranging from 0.05 nM to 10 nM. The pink contour in each graph represents the uninduced population.
We did not observe a graded response in eYFP production in response to the sweep of E2 induction, instead observing saturation at 0.25 nM E2. We hypothesize that, because MCF7 overexpresses the estrogen receptor, relatively small E2 signals can still be transduced to large responses.
Conclusions: Our promoters were able to successfully sense changes in estrogen signaling in the MCF7 cell line. All three promoters demonstrate a fold increase of similar magnitude upon exposure to estrogen. We have not yet been able to demonstrate a graded response of our promoters to changing E2 levels in MCF7. Instead we observed saturation at our lowest concentration tested, .25 nM.
Sensing Estrogen Signaling in ISH
Testing On - Off Functionality of Estrogen Sensitive Promoters
Finer Characterization Studies
In our next set of experiments, we sought to obtain a finer characterization of our promoters through exposing transfected cells to a sweep of E2 concentrations including .05 nM, 0.1 nM, 0.25 nM, 0.5 nM, 1 nM, 2.5 nM, 5 nM, 10 nM. We had hypothesized that our promoters would demonstrate a graded response to
