I have generated an extremely sensitive reporter cell line that unveils a new domain of Hex expression in murine embryonic stem (ES) cells. the introduction of a cDNA encoding a tagged version of Hex upstream of an internal ribosomal entry site and a variant of the Yellow Fluorescent Protein, Venus, into the first exon of <i>Hex</i>, has revealed a heterogeneous expression pattern among ES cell cultures. Manipulation of fibroblast growth factor signalling alters the percentage of venus positive cells and suggests that this subpopulation maybe failed to become endoderm, the earliest domain of <i>Hex </i>expression in the mouse embryo. Although there is an equivalence of <i>Oct3/4 </i>in both venus positive and negative subpopulations, <i>Nanog </i>and venus appear mutually exclusive. Microarray and quantitative PCR analyses show an enrichment of primitive endoderm specific genes in venus positive ES cells while markers of pluripotency are comparatively reduced. While clonal density plating of these subpopulations demonstrate interconvertability, venus positive ES cells have a reduced ability for clonal growth and contribution to the embryo in chimera analyses. I also attempt to overexpress <i>Hex </i>in ES cell cultures. Establishment of stable clones overexpressing <i>Hex </i>in ES and other cell types is difficult, suggesting intolerance. Construction of an inducible system to characterize the phenotype of ectopic <i>Hex </i>expression in ES cells reveals the onset of apoptosis upon induction by a mechanism that depends on its ability to bind DNA. These observations reflect previous studies which suggest that <i>Hex </i>is a key regulator in maintaining a balance between immediate early cell lineage decisions and proliferation. These studies suggest that <i>Hex </i>may be an important marker of early cell fate decisions, but is probably not the primary mediator of early blastocyst/ES cell asymmetry.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:642576 |
| Date | January 2008 |
| Creators | Canham, Maurice A. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/13303 |
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