Week 8

                This week was spent planning a second experiment that is highly related to the other, longer term experiment that we are still working on. The question we hope to answer with this experiment is, “Is there a threshold to regulate different cell fates? In other words, do different quantities of E2F1 cause cells to enter quiescence, proliferation, arrest, or apoptosis?” In order to answer this question, we are going to take advantage of the fact that the transient cells that we currently have made using the same plasmid contain 10-100 fold variations in quantity of the plasmid. Transient cells are cells that are expressing the plasmid without having incorporated the plasmid into its own DNA, so that the color fades over the course of cell divisions. In most experiments, transient cells are a problem, and the stable cell line that we are trying to make would be used in its place.

                In this experiment, we are going to make use of flow cytometry. We will stain the DNA with a red fluorescent dye called 7-AD, and the protein E2F1 produces already fluoresces yellow with YFP. Flow cytometry will quantify the level that the cells are glowing, and by comparing the amount of DNA to the amount of E2F1, we can determine what quantities of E2F1 cause cells to divide, be arrested, or die.