UBC Theses and Dissertations
Development of embryonic stem cells expressing endogenous levels of a fluorescent protein fused to the telomere binding protein TRF1 Miller, Shelley Bonnie
Telomeres are the repetitive DNA sequence and associated proteins found at the ends of linear chromosomes. They have a role in biological processes including meiosis and aging as well as implications in a number of genomic instability disorders and cancers. Telomeres maintain genomic stability by protecting chromosome ends from terminal fusions and misidentification as DNA damage sites. Their wide range of functions has resulted in an increased interest in developing tools to study the dynamics of telomeres in live cells. To do this, current studies use the ubiquitously expressed protein Telomere Repeat Factor 1 (TRF1) tagged with a fluorescent protein. TRF1 is a negative regulator of telomere length that binds exclusively to telomere repeats. Over-expression of the fluorescent protein fused to TRF1 has been a useful tool to track telomere movement. The foci formed by the tagged TRF1 protein accurately represent the number of telomeres expected in the cells and the localization is maintained throughout the cell cycle. A caveat with this system is that over-expression of TRF1 leads to accelerated telomere shortening, as well as replication defects that can stall telomere replication. These caveats make it difficult to draw conclusions about telomere dynamics based solely on observations of cells over-expressing fluorescently tagged TRF1. To eliminate problems associated with protein over-expression, I have tried to develop knock-in embryonic stem (ES) cells expressing fluorescently tagged TRF1 from the endogenous Trf1 promoter. To do this, I have used a recombineering technique using Bacterial Artificial Chromosomes (BACs). BAC recombineering allows for the direct knock-in of a fluorescent tag into the mouse Trf1gene locus. Genetic constructs with the correct sequence inserts have been obtained and have been used for transfection of ES cells. While no correctly targeted ES cells have been identified so far, the expectation is that ES cell lines with correctly targeted fluorescently tagged TRF1 will be obtained in the near future. Such lines will be used to study telomere dynamics in ES cells, differentiated cells generated from ES cells, as well as to generate mice.
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