Tour-de-Force

Tour-de-Force: Interplay between Mitochondria and Cell Cycle Progression Fall 2007Proposed ResearchCell lines and CultureIn all of our experiments we will use mouse embryonic fibroblasts (MEFs). The cells will be isolated frommouse embryos and immortalized. Cell lines will be cultured at a temperature of 37 Celsius, at constantatmospheric PH; 95% air and 5% carbon dioxide. These cells are especially useful for our project becausethey are relatively inexpensive, un-differentiated and rapidly proliferating. We chose immortalized cellsbecause we need large quantities of cells, and would like to conserve the cell line throughout ourexperiments. Despite immortalization these cells have a normal cell cycle, maintain contact inhibition andcontain the vital cell cycle pathways (Cell Line Database). Cells will be grown in complete DMEM mediumcontaining 25mM of glucose, as well as 2 mM glutamine, 100 U/ml penicillin100, and 10% new born calfserum (Chen et al., 2006).To verify the information we obtain with the MEF cells, we will use a second cell line to repeat anyexperiments we deem critical at the end. We will use CHO-Chinese hamster ovary epithelial cells. Cellswill be supplied by the American Type Culture Collection (ATCC).Synchronizing cellsFor all our experiments we will need a cell culture of synchronized cells. In order to synchronize the cells,we will use mitotic shake-off (Appendix A). Mitotic Shake-off is a reliable method that starts thesynchronized cell cycle in G1, which is advantageous for us since we are interested in especially this partof the cell cycle. Cells will remain well synchronized for at least 1 cell cycle.Cell cycle length and G0 – determination of standards in our cell line0.1 How long is a normal cell cycle and how long are its phases?Since an important part of this research will focus on the cell cycle progression and cell cycle arrest, it isimportant to know the length of a cell cycle in our cell line. We will also determine the length of theseparate phases, so we can compare the length of G1 in this experiment with the possibly prolonged G1in experiment 2. We expect the cell cycle to be approximately 24 hours (Norbury & Nurse, 1992), thereforewe will measure for 30 hours.Experiment 0.1For this experiment we will take cells that are grown on a complete medium and have not been subjectedto any experiments, measurements, or other intervening conditions. The cell culture will be synchronizedand divided up in 60 small samples. Every 30 minutes a sample will be used to determine the phase thecells are in. The cells will be fixed and permeabilized. The cell phase will be determined by the “Cell phasedetermination Kit” from Cayman Chemicals (Appendix A) according to the protocol. By propidium iodidestaining, the DNA content of the cell can be determined with a flow cytometer, which allows us todetermine the percentage of cells in a sample that are in G0/G1, G2 or S phase. M phase will becalculated.We will start measuring G1 as soon as over 50% of the cells are in this phase. We willsubsequently measure one of the samples every 30 minutes and determine the phase according to thephase >50% of the cells are in. The length of M-phase will be determined when the first cycle is almostcompleted, because with mitotic shake-off, we do not know where in M phase the cells actually start. Thelength of M will be determined by the length of the total cell cycle (determined when over 50% of the cellsare in G1 for the second time) minus the length of the other phases.0.2 What are the physiological concentrations of ATP, Cyclin A, B and D1,phosphorylated/unphosphorylated AMPK and phosphorylated/unphosphorylated Rb throughout the cellcycle?These protein levels are of importance for the following experiments, therefore a reference of thephysiological conditions is required for the specific cell line we use.SCI 332 Advanced Molecular Cell Biology Research Proposal 43