Loss of Cul3 in Primary Fibroblasts
DNA holds the essence of life, and interpreting it can give us clues about cell dynamics. Using retrospective analysis of cell processes, such as protein production and degradation, we try to answer some basic questions, taking advantage of cells being the fundamental units of life. Disruption of these complex processes results in abnormality, dysfunction, and eventually cell death. Cul3 is an essential enzyme that regulates the levels of proteins that in turn regulate transitions between different cell cycle stages. Cell cycle is a description of progressive order of events that lead to cell division. When the Cul3 gene is knocked out in cells of living organisms, some cells undergo apoptosis, or programmed cell death. This research focuses on the role of Cul3 in regulation of cell cycle transitions with a particular interest in how cells that lose Cul3 enter into the apoptotic pathway. The experimental system utilizes a special allele of Cul3 that was constructed in the lab and contains flanking DNA signals for a recombinase called Cre. When expressed, the Cre recombinase knocks out the Cul3 gene. Cells that have lost the gene are then monitored and analyzed for DNA breaks. We introduced the Cre gene using retroviral infection of primary fibroblasts containing the Cul3 special allele. This virus also expresses a special form of green fluorescent protein that allows us to identify cells that lost the Cul3 gene. As part of the experimental design, controls, which are cells that cannot delete the Cul3 gene and viruses that only express the green indicator protein, are included.
Faculty Mentor: Jeffrey Singer