Search

This thesis describes the optimisation and implementation of a conditional transgene expression system in mouse embryonic stem (ES) cells for the investigation of signalling pathways controlling in-vitro differentiation. This system, the tetracycline-induced conditional gene expression system (tet-system) allows exquisite control over the transgene expression in vitro and in vivo. The system itself is comprised of two components, a transactivator gene, and a transactivator-response element (TRE) which is linked to the transgene to be regulated. The transactivator gene encodes a fusion protein consisting of a modified tetracycline repressor and transcriptional activation domain moieties, and is constitutively expressed. In the presence of doxycyline (dox) the transactivator protein binds to the TRE sequence, which itself consists of an array of operator sequences from the tetracycline operon linked to a minimal promoter region. Transcription of the transgene consequently ensues. This version of the tet-system is referred to as the tet-on system. In order to establish high and homogenous expression of inducible transgenes in Es cells using the tet-on system, the utilisation of different promoters to direct expression of the transcriptor component (rtTA2s-M2) was investigated, as was the use of different loci at which to target the system components. It was found that the promoter sequence CAG, when linked to the transactivator rtTA2s-M2 sequence and targeted to the 5’ region of the housekeeping gene hypoxantine ribosyltransferase (hprt)gave rise to homogenous expression of the transactivator. However upon targeting the 3’ region of hprt with the TRE and a linked DsRED2 reporter transgene, addition of dox resulted in heterogenous expression of the transgene. In an alternative strategy, cells expressing the rtTA2s-M2 transactivator from the endogenous ROSA26 promoter (R26rtTA2s-M21F cells), were targeted to integrate the TRE and linked DsRED2 reporter to the 5’region of hprt. In cell lines harbouring both components of the tet-system, addition of dox resulted in high and homogenous expression of the DsRED2 transgene.