These Rosa26PA-Cre A20 &ldquo;all-in-one&rdquo; mice contain a photoactivatable Cre recombinase (PA-Cre 3.0) that allows for highly efficient DNA recombination in an internal organ of living mice through noninvasive external illumination using blue light-inducible dimerization of the Magnets-opti system. Specifically, PA-Cre 3.0 is floxed and consists of a split Cre (59/60) each complemented with nMag or pMag (respectively), separated by P2A self-cleaving peptide sequence (2A), and a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) followed by a polyadenylation (pA) site. This cassette is preceded by a frt-flanked STOP cassette and followed by a nuclear-localized red fluorescent protein (mKate2) and another pA site. The above mentioned cassettes were placed under control of the CAG (CMV enhancer/chicken beta-actin) promoter from the endogenous Gt(ROSA)26Sor locus for widespread expression. The frt-flanked STOP cassette (containing a neo cassette and a pA site) prevents PA-Cre 3.0 and mKate2 expression in the absence of FLP recombinase. After removal of the STOP cassette, blue light dimerization of the Magnets-opti system results in reassembly of split PA-Cre 3.0 fragments in flp-expressing tissues. The floxed PA-Cre 3.0 is then removed by itself, leaving only the mKate2 reporter expression. mKate2 expression is evident, and thus recombination is successful, after blue-light illumination, when these &ldquo;all-in-one&rdquo; PA-Cre 3.0 A20 mice are bred to flp-expressing mice or when tissues are infected with Flp lentiviruses without unintended leakiness in the dark condition, in contrast to previous versions of the PA-Cre system. Heterozygous mice are viable and fertile. The donating investigator has not attempted to make this strain homozygous (November 2020). Of note, Rosa26PA-Cre B4 mice (Stock No. <a href="https://www.jax.org/strain/033545">033545</a>) are a similar strain that contains a frt-flanked STOP cassette upstream of PA-Cre 3.0, allowing for tissue specific expression of reassembly of split PA-Cre 3.0 fragments, and expression of PA-Cre 3.0 following FLP-frt recombination.

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Rosa26PA-Cre A20 mice express a photoactivatable Cre recombinase (PA-Cre 3.0) following exposure to FLP recombinase. Specifically, FLP-mediated removal of the frt-flanked STOP cassette results in expression of the individual split Cre segments (nMag::CreN59 and pMag::CreC60). Subsequent illumination with blue-light causes nMag-pMag dimerization (Magnets-opti dimerization), which brings the split Cre segments together to form a functional Cre recombinase molecule. This allows for highly efficient DNA recombination in an internal organ of living mice through noninvasive external blue light illumination, without unintended leakiness in the dark condition. Additionally, Rosa26PA-Cre A20 has loxP-sites flanking the PA-Cre 3.0 cassette, which is followed by a nuclear-localized red fluorescent protein (mKate2). As such, after FLP exposure and blue light illumination, the PA-Cre 3.0 removes itself - resulting in mKate2 fluorescence. Of note, the donating investigator also has available Rosa26PA-Cre B4 mice (Stock No. <a href="https://www.jax.org/strain/033545">033545</a>) - a similar strain that contains a frt-flanked STOP cassette upstream of a PA-Cre 3.0, but the PA-Cre 3.0 is not flanked by loxP sites and does not have the mKate2 sequences.

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B6;129S-Gt(ROSA)26Sor<tm1(CAG-Ncre/vvd*,vvd*/Ccre,-mKate2)Yz