Fig. 6. Generation and validation of all-in-one version of PA-Cre 3.0 mouse line.

a Schematic representation of PA-Cre 3.0 “All-in-One” mice targeting strategy. CAG promoter and PA-Cre 3.0 knocked-in to Gt(ROSA)26Sor (Rosa26) locus was conducted together with stop cassette inserted between FRT cassettes and nuclear-localizing signal (NLS)-fused mKate2 red fluorescent protein. The PA-Cre 3.0 is located between loxP pair. Flp-mediated excision of the FRT-flanked stop cassette results in the expression of PA-Cre 3.0. After the blue-light stimulation and PA-Cre activation, the PA-Cre is self-cleaved. PA-Cre-mediated excision of the PA-Cre 3.0 cassette including WPRE element induces NLS-mKate2 expression. The mKate2 red fluorescence signals located in nuclei can be used as a Cre-loxP recombination reporter. b Schematic representation of experimental time course for MEFs derived from Rosa26^{PACre A20/FLPe} mice. The cells were illuminated for 6 h (470 ± 20 nm, 2 W/m², 6 h continuous). The cells were imaged 42 h after illumination. c Representative mKate2 fluorescence images of PA-Cre 3.0 A20 MEFs (Left images, the bright field and right images, the mKate2 red fluorescence; n = 3, Scale bar: 100 μm). d Schematic representation of experimental time course for cortical neurons derived from Rosa26^{PACre A20/WT} mouse brains with illumination. The CAG-Flpe lentivirus was infected at serial 2 days. At the following day (day 3), the cells were illuminated for 24 h (470 ± 20 nm, 1 W/m², 24 h continuous). e Representative mKate2 fluorescence images of PA-Cre 3.0 A20 neurons (Left images, the bright field and right images, the mKate2 red fluorescence; n = 3, Scale bar: 100 μm).