VEZF1 is a highly conserved vertebrate transcription factor that is essential for mammalian development. The gene regulatory functions of VEZF1 are largely undetermined. The generation of human cells depleted or absent of VEZF1 would greatly assist the study of VEZF1 functions and mechanism of action. This study makes use of synthetic biology technologies to either repress or knock out VEZF1 gene transcription to enable further studies of VEZF1 function. This study explores various strategies to use engineered DNA-binding proteins to direct the repression or mutation of a gene of interest. Zinc Finger (ZF) and Transcription Activator Like Effector (TALE) proteins that specifically recognise DNA sequences at the VEZF1 gene promoter were constructed using modular or Golden Gate assembly methods. The ability of TALE fusion proteins to function in human cells was studied. An expression vector system was created to assemble TALE Repressor (TALER) fusion proteins. The use of TALERs allowed for the rapid assessment of TALE protein binding at their chromosomal targets in human cells. Transient expression of most of the assembled TALE repressor proteins resulted in reduced VEZF1 transcription. A subset resulted in very substantial VEZF1 repression, making them useful tools for the study of VEZF1 function. Functional TALE domains were assembled into TALE nuclease (TALEN) fusion proteins. TALEN expression vectors were developed to assemble TALEN proteins with optimised expression, cleavage activity and target specificity. Transient expression of TALEN proteins in human cells was used to direct the cleavage and error-prone DNA repair of the VEZF1 promoter. Following development of the assays used to detect TALEN-directed mutations, several functional TALEN pairs were identified. Some TALENs resulted in over 65% mutation rates, with some mutations removing the VEZF1 promoter. These TALENs will be useful for the development of VEZF1 knock out cell lines. Interestingly, our study reveals a correlation between TALE length and the activity of TALERs and TALENs that should be considered in the future application of TALE proteins.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:634007 |
| Date | January 2015 |
| Creators | Gourlay, Elaine Margaret |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/5875/ |
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