title: Efficient ROSA26-based conditional and/or inducible transgenesis using RMCE-compatible F1 hybrid mouse embryonic stem cells.
abstract: The conditional Cre/loxP system and/or the doxycycline (Dox) inducible Tet-on/off system are widely used in mouse transgenesis but often require time consuming, inefficient cloning/screening steps and extensive mouse breeding strategies. We have therefore developed a highly efficient Gateway- and recombinase-mediated cassette exchange (RMCE)-compatible system to target conditional and/or inducible constructs to the ROSA26 locus of F1 hybrid Bl6/129 ESCs, called G4 ROSALUC ESCs. By combining the Cre/loxP system with or without the inducible Tet-on system using Gateway cloning, we can rapidly generate spatial and/or temporal controllable gain-of-function constructs that can be targeted to the RMCE-compatible ROSA26 locus of the G4 ROSALUC ESCs with efficiencies close to 100 %. These novel ESC-based technologies allow for the creation of multiple gain-of-function conditional and/or inducible transgenic ESC clones and mouse lines in a highly efficient and locus specific manner. Importantly, incorporating insulator sequences into the Dox-inducible vector system resulted in robust, stable transgene expression in undifferentiated ESCs but could not fully overcome transgene mosaicism in the differentiated state.
authors.affiliation: VIB Department for Molecular Biomedical Research, Vascular Cell Biology Unit, Ghent, Belgium.
authors.forename: Lieven
authors.initials: L
authors.lastname: Haenebalcke
authors.forename: Steven
authors.initials: S
authors.lastname: Goossens
authors.forename: Michael
authors.initials: M
authors.lastname: Naessens
authors.forename: Natascha
authors.initials: N
authors.lastname: Kruse
authors.forename: Morvarid
authors.initials: M
authors.lastname: Farhang Ghahremani
authors.forename: Sonia
authors.initials: S
authors.lastname: Bartunkova
authors.forename: Katharina
authors.initials: K
authors.lastname: Haigh
authors.forename: Tim
authors.initials: T
authors.lastname: Pieters
authors.forename: Pieterjan
authors.initials: P
authors.lastname: Dierickx
authors.forename: Benjamin
authors.initials: B
authors.lastname: Drogat
authors.forename: Omar
authors.initials: O
authors.lastname: Nyabi
authors.forename: Dagmar
authors.initials: D
authors.lastname: Wirth
authors.forename: Jody J
authors.initials: JJ
authors.lastname: Haigh
mesh_terms: D000818:Animals; D003433:Crosses, Genetic; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D018014:Gene Transfer Techniques; D056426:Genetic Loci; D006824:Hybridization, Genetic; D008297:Male; D051379:Mice; D022661:RNA, Untranslated / Q000378:metabolism*; D045522:Recombinases / Q000378:metabolism
chemical_list: C110853:Gt(ROSA)26Sor non-coding RNA, mouse; D022661:RNA, Untranslated; D045522:Recombinases