Conjugation of the Human Immunodeficiency Virus Transactivator of Transcription (TAT) to active proteins allows transport into the intracellular environment. This feature can be harnessed to deliver combinations of reprogramming factors (RFs) such as c-Myc, Oct4, Klf4 and Sox2 into somatic cells to derive induced pluripotent stem cells (iPSCs). For this project, TAT-fusion proteins including four TAT-conjugated RFs (TAT-RFs) have been produced and purified. All four TAT-RFs can bind specific DNA sequences. Bioactivity was tested in live cells using a novel assay based on an engineered fibroblast cell line that can be induced to express RFs by doxycycline and subsequently generate iPSCs. To test each TAT-RF, reprogramming was blocked by transient silencing of a single RF by siRNA and rescued by the corresponding TAT-RF. The results of this assay suggested that TAT-Klf4 was bioactive in cells; however, definitive evidence could not be obtained for other RFs.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/31344 |
| Date | 14 December 2011 |
| Creators | Moghaddam, Bahar |
| Contributors | Audet, Julie, Zandstra, Peter W. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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