Team:MIT/Experiments/Promoters/Experiment-Details

Promoter Characterization Experiments in MCF-7, ISH, and tHESC

Promoter Testing Workflow

Our synthetic mammalian 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.


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 coordinatedwith the Griffith Lab at MIT affiliated with the Center for Gynepathology Research at MIT.


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.

Finer Characterization Studies

pERE3

Fluorescent reporter for transfection vs. reporter for promoter activity

The induced MCF-7 cells show up to a 10 fold difference in eYFP fluorescent output compared with the uninduced cells. However, there is not a defined fold difference in fluorescent output between the varying concentrations of estrogen induced.



Bar Graph Comparing Fluorescent eYFP Reporter at Varying Concentrations of Estrogen

The concentration of estrogen that stimulated the highest level of promoter activity was 0.05 nM, whereas the concentration that stimulated the lowest level of activity was 0.25 nM. These results are inconsistent with the hypothesis that increasing estrogen concentration in the cells will increase promoter activity, especially because the results from the 0.05 nM E2 well and the 10 nM E2 well are just about equal. Through a little research, we've concluded that the lack of staggered results is due to the fact that we dissolved E2 in ethanol. According to Etique et. al [1], MCF-7 cells grown in an ethanol medium were correlated with increased proliferation, ERalpha content, and ER transcriptional activity. We tweaked our experimental design for future experiments to contain a vehicle control which accounts for the proliferation and increase in ERalpha caused by ethanol. Then we can more accurately compare our promoters' basal levels with induced levels.

pERE5

The results show about a 2- to 4-fold increase in promoter activity when induced with estrogen. However, when comparing the estrogen fine sweep results, it appears that the lower concentrations of estrogen (0.05 nM, 0.25 nM) had increased fluorescent output levels from the promoter compared with the cells that were induced with higher concentration (5 nM, 10 nM). The promoter proves to be functionable, but not necessarily to the caliber we need for our project. Our project relies on estrogen responsive promoters that can sufficiently detect the difference between the phases of the menstrual cycle. A two to four fold increase in promoter activity and an illogical correlation between output and varying estrogen levels will not be very helpful in the context of our circuit. So, we must keep trying.

pERE6

The results show about a 2 fold difference between basal and induced promoter activity, with no evident correlation between output and increasing concentration of E2 in the cell. Similarly to the pERE5 promoter, the fold difference between the on/off states of the promoter might not be sufficient for our circuit to identify different phases of the menstrual cycle.

Notes of Interest

-In this experiment, our control was an uninduced well of cells transfected with the promoter-reporter construct. There was no vehicle control. Cell proliferation might partially be the cause increased fluorescent output.

-Transfection Efficiency is around 10-15%.

Overall Conclusion

The pERE3 promoter showed the greatest difference between off/on states out of all three promoters tested in MCF-7 cells. Based on these experimental results, we would choose the pERE3 promoter as the estrogen-responsive for our circuit. However, we still need to test these promoters in endometrial cell lines to best model conditions our circuit will be implemented in, and use a vehicle control to account for cell proliferation and greater ERalpha expression. This experiment was a good baseline test to see how our promoters worked, and we used it as a stepping stone to design future experiments and compare results with.