Studying the Role of Condensin Protein in Mitosis
Jake Heier, Miranda LaPointe (Dr. James Paulson)
Condensin is in the family of structural maintenance of chromosome (SMC) proteins and is believed to play a role in stabilizing the condensed structure of mitotic chromosomes. We would like to learn more about its role and to do so we plan to use a chicken DT40 cell line that has a conditional knockout of the condensin gene. Our goal is to isolate mitotic chromosomes from cells with diminished amounts of condensin and then study their structure by electron microscopy.
As a prerequisite to this approach, several methods needed to be tested and analyzed. In the summer of 2009, we developed methods for visualizing DNA by electron microscopy using the materials we had available. We used DNA plasmids to work out methods for spreading DNA with cytochrome c, staining with uranyl acetate and shadowing with platinum. These techniques will later be used for electron microscopy of histone-depleted chromosomes. We also optimized methods for synchronizing DT40 cells in mitosis and quantifying the percentage of cells in mitosis. These procedures will be essential in future work when we plan to isolate and study the structure of mitotic chromosomes from cells depleted of condensin.