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- W3047162439 abstract "Transgenic mice are often generated by Random Integration (RI) of the transgene into the genome of murine Embryonic Stem (mES) cells. Since the chromosomal integration site of the gene of interest has a strong impact on expression patterns in the animal (oposition effecto), generation of transgenic mouse models is hindered by high screening efforts to identify appropriate loci. Strategies devised to circumvent the problems of RI, e.g. site specific integration by homologous recombination, suffer from various limitations like the small number of suitable integration sites currently available. Hence, the aim of this work was to screen for chromosomal loci in mES cells that allow for high and well regulated transgene expression in mice and to render them reusable via Recombinase Mediated Cassette Exchange (RMCE). The screening cassette applied for this purpose contained an autoregulatory positive feedback loop driven by the tetracycline dependent system. As autoregulated transgene expression has not yet been examined in detail on transgenic mouse level, evaluation of this mode of regulated expression was a major issue of this work. Luciferase reporter levels from two random loci and the ROSA26 reference locus revealed strong variations that seem to be specific to autoregulation. In vivo bioluminescence analyses of transgenic mice derived from the two above mentioned clones showed a strong heterogeneity of expression between individuals of the same line with some animals completely shutting off expression. As expected for the autoregulated design, luciferase expression in one transgenic mouse line could only be induced by doxycycline if the animal had previously shown detectable basal levels. For both transgenic lines activity of the cassette was restricted to certain tissues. In conclusion, proof of principle for the envisioned tag-and-target strategy has been provided by this work and the tools and methods have been established to start large scale screening." @default.
- W3047162439 created "2020-08-10" @default.
- W3047162439 creator A5013064739 @default.
- W3047162439 date "2008-02-28" @default.
- W3047162439 modified "2023-09-23" @default.
- W3047162439 title "Establishing murine embryonic stem cells for generation of transgenic mice featuring predictable and regulatable transgene expression" @default.
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