Tet ON/OFF inducible gene expression and homologus chromosme inactivation in ES cells and tetraploid mice
Anton WUTZ, IMP Email: , Wutz Lab Website
Collaboration: Erwin WAGNER, Josef PENNINGER
An inducible gene expression system has been developed for ES cells. This system is based on expression of the tetracycline dependent transcriptional activator under the control of the ROSA26 locus, which is active in almost all embryonic cells. The aim of this GEN-AU subproject is to introduce this tetracycline regulated expression system into our newly derived F1 "tetraploid" ES cells, which can be used to generate ES mice by injection into tetraploid blastocysts. Such cells can then be used to drive expression of selected transgenes in ES mice by induction with doxycycline. Using this system we plan to study the effect of chromatin regulators on development and disease.
In addition, using transgenes derived from sequences of the mouse Xist RNA we have been able to induce chromosome wide gene repression on autosomes in ES cells . This provides a possible route to circumvent masking of mutations by the heterozygous state in screens. Indeed, a major problem in mammalian genetics is the lack of efficient screening technology. We will aim at developing a novel approach combining inducible Xist expressing ES cells with tetraploid technology for genetic screens in mouse tissues. To achieve this we aim at the generation of inducible Xist transgenic ES cell lines for tetraploids. The insertion sites will be mapped to autosomes by DNA FISH analysis and a panel of selected insertions will be used to generate ES mice. If successful such ES cells can potentially be used for large insertional mutagenesis and phenotypes can be unmasked by inactivating the homologous chromosome. Furthermore, such mice if viable will be used by the Wutz group to study the consequences of large genomic imbalances in gene dosage as well as the mechanism of mammalian X-inactivation.
