Visualizing Protein Interactions in the Live Mouse Pituitary Cell Using Wide-Field FRET Microscopy

To explore the interaction of Pit-1β with the prolactin transcription factors C/EBPα and Pit-1, GHFT1-5 mouse pituitary cells were transfected with EGFP and mOrange-fused proteins in an effort to observe and quantify FRET using wide-field microscopy and image processing using an algorithm. The data rendered inconclusive results, as FRET signals were insufficiently low. However, the problems can be remedied by altering the quantity of DNA transfected so as to increase the probability that the acceptor molecules will be in the appropriate orientation for FRET to occur. It is suitable to conclude that FRET microscopy is the technique of choice in visualizing the interaction of Pit-1β, C/EBPα, and Pit-1 in living pituitary cells, regardless of the less than ideal pair of EGFP and mOrange as donor and acceptor fluorophores, respectively.