<p>Recent advances in gene targeting technology have significantly shaped modern-day mouse genetics, as they allow for the accurate analysis of gene function <em>in vivo</em>. By capitalizing on conventional methodologies that are based on homologous recombination, the advent of artificially engineered nucleases, like transcription activator-like effector nucleases (TALENs), enables precise genome editing without the need for conventional targeting vectors, which typically possess long “arms” of homology that are difficult to work with, even with recombineering strategies employing bacterial artificial chromosomes. Unlike traditional techniques, these novel nucleases can be engineered in less than a week and together with compact targeting vectors, can be used to easily manipulate almost any locus in the mouse genome.</p> <p>The current selection of commercially available antibodies makes it difficult to assess the specific roles of protein isoforms during early development. The Tcf/Lef family of transcription factors comprise of key downstream effector proteins of the canonical Wnt/β-catenin signal transduction cascade. This pathway is implicated in the regulation of self-renewal and is dysregulated in a number of human diseases including cancers. Among the Tcf/Lef factors, Tcf3 has been heavily studied in mouse embryonic stem cells, due at least in part to the observation that its transcript levels are expressed at the highest levels compared to the others. Recently, it was proposed that a switch takes place between a repressive state mediated by Tcf3 to an activating β-catenin-Tcf1 complex in response to Wnt signals. Here, we use TALEN technology to introduce an epitope tag at the endogenous locus of <em>mTcf7</em>, the gene encoding the Tcf1 protein. By tagging the N-terminus of full-length and N-terminally truncated dominant-negative variants of Tcf1, we establish a tool to better study a previously unappreciated role for Tcf1 in regulating embryonic stem cell self-renewal and differentiation. Furthermore, we also show that the tagged variants generated exhibit similar protein expression levels to those of wild-type controls, and display nuclear localization as expected.</p> / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/15275 |
| Date | 31 August 2014 |
| Creators | Mahendram, Sujeivan |
| Contributors | Doble, Bradley W., Kristin Hope, Darren Bridgewater, Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Detected Language | English |
| Type | thesis |
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