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

Area of Science:

• Genetics and Genomics
• Transgenic Technology
• Stem Cell Biology

Background:

• Conditional and inducible gene expression systems (Cre/loxP, Tet-on/off) are crucial for mouse transgenesis.
• Traditional methods involve time-consuming cloning, screening, and complex breeding strategies.
• Targeting specific loci like ROSA26 is desirable for controlled gene expression.

Purpose of the Study:

• To develop a highly efficient system for generating conditional and/or inducible transgenic mouse models.
• To streamline the creation of spatial and temporal controllable gain-of-function constructs.
• To enable locus-specific targeting to the ROSA26 locus in embryonic stem cells (ESCs).

Main Methods:

• Development of a Gateway- and recombinase-mediated cassette exchange (RMCE)-compatible system.
• Integration of Cre/loxP and/or doxycycline (Dox)-inducible Tet-on systems using Gateway cloning.
• Targeting constructs to the ROSA26 locus in F1 hybrid Bl6/129 ESCs (G4 ROSALUC ESCs).
• Incorporation of insulator sequences into the Dox-inducible vector.

Main Results:

• Achieved near 100% efficiency in targeting conditional and/or inducible constructs to the ROSA26 locus.
• Rapidly generated multiple gain-of-function conditional and/or inducible transgenic ESC clones and mouse lines.
• Demonstrated robust and stable transgene expression in undifferentiated ESCs using insulator sequences.
• Observed that insulator sequences could not fully overcome transgene mosaicism in differentiated cells.

Conclusions:

• The novel Gateway/RMCE-based system significantly enhances the efficiency and speed of generating conditional and inducible transgenic mouse models.
• This technology facilitates locus-specific targeting to ROSA26, enabling precise control over gene expression.
• While effective in ESCs, further optimization is needed to address transgene mosaicism in differentiated states.