Efficient ROSA26-based conditional and/or inducible transgenesis using RMCE-compatible F1 hybrid mouse embryonic stem cells

Lieven Haenebalcke, Steven Goossens, Michael Naessens, Natascha Kruse, Morvarid Farhang Ghahremani, Sonia Bartunkova, Katharina Haigh, Tim Pieters, Pieterjan Dierickx, Benjamin Drogat, Omar Nyabi, Dagmar Wirth, Jody Jonathan Haigh

Research output: Contribution to journalArticleResearchpeer-review

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.
Original languageEnglish
Pages (from-to)774-785
Number of pages12
JournalStem Cell Reviews
Volume9
Issue number6
DOIs
Publication statusPublished - 2013

Cite this

Haenebalcke, L., Goossens, S., Naessens, M., Kruse, N., Ghahremani, M. F., Bartunkova, S., ... Haigh, J. J. (2013). Efficient ROSA26-based conditional and/or inducible transgenesis using RMCE-compatible F1 hybrid mouse embryonic stem cells. Stem Cell Reviews, 9(6), 774-785. https://doi.org/10.1007/s12015-013-9458-z
Haenebalcke, Lieven ; Goossens, Steven ; Naessens, Michael ; Kruse, Natascha ; Ghahremani, Morvarid Farhang ; Bartunkova, Sonia ; Haigh, Katharina ; Pieters, Tim ; Dierickx, Pieterjan ; Drogat, Benjamin ; Nyabi, Omar ; Wirth, Dagmar ; Haigh, Jody Jonathan. / Efficient ROSA26-based conditional and/or inducible transgenesis using RMCE-compatible F1 hybrid mouse embryonic stem cells. In: Stem Cell Reviews. 2013 ; Vol. 9, No. 6. pp. 774-785.
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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.",
author = "Lieven Haenebalcke and Steven Goossens and Michael Naessens and Natascha Kruse and Ghahremani, {Morvarid Farhang} and Sonia Bartunkova and Katharina Haigh and Tim Pieters and Pieterjan Dierickx and Benjamin Drogat and Omar Nyabi and Dagmar Wirth and Haigh, {Jody Jonathan}",
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Haenebalcke, L, Goossens, S, Naessens, M, Kruse, N, Ghahremani, MF, Bartunkova, S, Haigh, K, Pieters, T, Dierickx, P, Drogat, B, Nyabi, O, Wirth, D & Haigh, JJ 2013, 'Efficient ROSA26-based conditional and/or inducible transgenesis using RMCE-compatible F1 hybrid mouse embryonic stem cells' Stem Cell Reviews, vol. 9, no. 6, pp. 774-785. https://doi.org/10.1007/s12015-013-9458-z

Efficient ROSA26-based conditional and/or inducible transgenesis using RMCE-compatible F1 hybrid mouse embryonic stem cells. / Haenebalcke, Lieven; Goossens, Steven; Naessens, Michael; Kruse, Natascha; Ghahremani, Morvarid Farhang; Bartunkova, Sonia; Haigh, Katharina; Pieters, Tim; Dierickx, Pieterjan; Drogat, Benjamin; Nyabi, Omar; Wirth, Dagmar; Haigh, Jody Jonathan.

In: Stem Cell Reviews, Vol. 9, No. 6, 2013, p. 774-785.

Research output: Contribution to journalArticleResearchpeer-review

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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

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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.

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