TY - JOUR
T1 - Efficient ROSA26-based conditional and/or inducible transgenesis using RMCE-compatible F1 hybrid mouse embryonic stem cells
AU - Haenebalcke, Lieven
AU - Goossens, Steven
AU - Naessens, Michael
AU - Kruse, Natascha
AU - Ghahremani, Morvarid Farhang
AU - Bartunkova, Sonia
AU - Haigh, Katharina
AU - Pieters, Tim
AU - Dierickx, Pieterjan
AU - Drogat, Benjamin
AU - Nyabi, Omar
AU - Wirth, Dagmar
AU - Haigh, Jody Jonathan
PY - 2013
Y1 - 2013
N2 - 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.
AB - 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.
UR - http://download.springer.com/static/pdf/618/art%253A10.1007%252Fs12015-013-9458-z.pdf?auth66=1391316575_ad2006a746ea9c9942f1c447044cd4c9&ext=.pdf
U2 - 10.1007/s12015-013-9458-z
DO - 10.1007/s12015-013-9458-z
M3 - Article
VL - 9
SP - 774
EP - 785
JO - Stem Cell Reviews and Reports
JF - Stem Cell Reviews and Reports
SN - 2629-3269
IS - 6
ER -