Part:BBa_K2100009:Experience

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Applications of BBa_K2100009

This entry vector was recombined into an expression vector pPRE4:eYFP with an LR reaction. The construct was characterized in tHESC and MCF-7 cells.

We transfected pPRE4:eYFP into tHESC to test its on/off functionality by comparing cells induced with MPA and cells induced with 1 uM MPA. We transfected hEF1a:mKate into tHESC as well as a transfection marker. 250 ng of each construct was used, maintaining a 1:1 ratio of DNA.

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The results showed little to no increase in fold difference of yellow fluorescence when comparing induced vs. uninduced cells. Unfortunately, we could not prove with this experiment that the PRE3 promoter increases activity in response to progesterone induction.

Characterizing the PRE4 promoter in MCF-7 cells showed more promising functionality. We transfected pPRE4:eYFP and hEF1a:mKate as a transfection marker into MCF-7. 250 ng of each construct was used, maintaining a 1:1 ratio of DNA. Like the tHESC experiment, the cells were induced with 1 uM of MPA and compared with an uninduced control.

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. pPRE4 has approximately a five fold increase in activity when induced with MPA for cells with lower amounts of plasmid as seen by the large fold difference at lower levels of red florescence -- our transfection marker. We can believe that with a better transfection efficiency, the cells that do receive a large amount of plasmids will demonstrate a similar fold difference between on and off when induced with MPA.

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