A single ROSA26 mouse line for robust tdTomato labeling with Cre, FLP, Dre, Vika, SCre, and VCre recombinases.

Maintaining separate reporter lines for diverse site-specific recombinases (SSRs) imposes a major logistical bottleneck in complex genetic modeling. Here, we developed the ROSA26-6R reporter mouse, a single, universal line providing robust tdTomato expression for six distinct SSRs: Cre, FLP, Dre, Vika, SCre, and VCre. By crossing this line with newly generated CAG-driven SSR driver lines, we established the first systematic in vivo recombination profile for these systems, revealing high recognition specificity for each SSR and identifying Vika and VCre as highly efficient systems comparable to the gold-standard Cre/loxP. The bright native fluorescence in this line enables high-resolution, antibody-free visualization of fine structures, including dendritic spines and axonal trajectories, and facilitates whole-embryo 3D imaging of the developing nervous system. By consolidating responsivity to six SSRs into a single genomic locus, the ROSA26-6R mouse significantly reduces colony maintenance burdens, while providing a versatile, high-performance platform for multi-feature genetic labeling and advanced imaging.