In order to study the behaviour and normal potential of axial progenitors and their differentiated descendants, transgenic mice carrying a 4-hydroxytamoxifen (4OHT) inducible form of <i>cre </i>recombinase, under the control of a fragment of the <i>T (Brachyury)</i> regulatory sequence known to confer expression in the primitive streak and tail bud, but not in the node or notochord, was generated. On crossing these ‘TcreER<sup>T2</sup>, mice with a Cre-dependent lacZ reporter line, embryos carrying both transgenes exhibited inducible primitive streak/tail bud-specific Cre activity upon administration of 4OHT to pregnant mothers. In the absence of 4OHT, Cre activity was not detected. Labelled descendants of streak cells populated somites, lateral/intermediate mesoderm and neurectoderm. The tetracycline-controlled transcriptional activation system (tet-on) was developed and tested as a means to reversibly manipulate gene function in axial progenitors. The reverse tetracycline transactivator (rtTA) was placed under the control of the primitive streak/tail bud-specific <i>T</i> promoter. T-rtTA was targeted to the Rosa26 locus in ES cells to provide a stable transcriptionally active genomic environment, and its function tested by inducing expression of a DsRed reporter gene from the tetracycline response element (TRE), which was integrated at the hprt locus. After allowing the cells to differentiate in the presence of doxycycline, induction was detected by fluorescence microscopy, FACS analysis and RT-PCR. However, DsRed levels were very low compared to a T-rtTA cell line generated in parallel in the lab by random integration. Therefore, the tet system is usable for manipulation of gene expression, but targeted integration provides no benefits over random integration. Thirdly, whole-mount embryo culture-electroporation (WEC-EP) in embryos that were lineage marked upon expression of the electroporated construct, was developed as an alternative experimental approach.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:649503 |
| Date | January 2007 |
| Creators | Dewhurst, Robert |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/13638 |
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