Transgenic manipulation of exogenous and endogenous gene expression in human embryonic stem cells (hESCs) is a powerful approach to decipher the genetic pathways dictating their developmental fate. Presently used genetic tools face limitations including leakiness in inducibility of expression, epigenetic silencing in long-term cell culture, low genomic integration efficiencies, small genetic cargo limit and lack of high-throughput cloning capabilities. To overcome these limitations, I have constructed R4-Integrase and piggyBac transposon genetic vector systems for stable transgene overexpression and knockdown in hESCs. Preliminary functional testing of the piggyBac vector system in HEK 293T and hESCs demonstrated vector inducibility as well as successful overexpression and knockdown of pluripotency factor OCT4. Concurrently, a cost-effective and high efficiency method for chemical transfection of hESCs was developed. Exogenous overexpression and knockdown of transcription factors in hESCs will aid in the elucidation of gene regulatory networks controlling pluripotency and developmental fate.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/33596 |
| Date | 27 November 2012 |
| Creators | Yamarte, Cesar |
| Contributors | Stanford, William L. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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