Neuroscience Research 73 (2012) 85–91 Contents lists available at SciVerse ScienceDirect Neuroscience Research jo u r n al hom ep age: www.elsevier.com/locate/neures Technical note A multifunctional teal-fluorescent Rosa26 reporter mouse line for Cre- and Flp-mediated recombination Itaru Imayoshi a,b,c,∗ , Kyoko Hirano a,d , Masayuki Sakamoto a,d , Goichi Miyoshi e , Tetsuya Imura f , Satsuki Kitano a , Hitoshi Miyachi a , Ryoichiro Kageyama a,g a Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan b The Hakubi Center, Kyoto University, Kyoto 606-8507, Japan c Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology (PRESTO), Kyoto 606-8507, Japan d Kyoto University Graduate School of Biostudies, Kyoto 606-8502, Japan e Smilow Neuroscience Program and the Department of Cell Biology, New York University, New York, NY 10016, USA f Department of Pathology and Applied Neurobiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan g Japan Science and Technology Agency, Core Research for Evolutional Science and Technology (CREST), Kyoto 606-8507, Japan a r t i c l e i n f o Article history: Received 26 December 2011 Received in revised form 2 February 2012 Accepted 8 February 2012 Available online 17 February 2012 Keywords: Cre Flp mTFP1 Knock-in mice Rosa26 a b s t r a c t Reporters of Cre and/or Flp activity are important for defining the spatial and temporal extent of Cre/Flp- mediated recombination. Here, we describe R26-CAG-LF-mTFP1, a multifunctional fluorescent reporter mouse that strongly expresses mTFP1 (bright teal fluorescent protein) after Cre- and Flp-mediated recombination. To meet the need for single recombinase-mediated reporter expression, we generated derivatives of R26-CAG-LF-mTFP1. The germline excision of the Frt-flanked stop cassette in R26-CAG-LF- mTFP1 generated a Cre-dependent reporter (R26-CAG-LoxP-mTFP1). Similarly, R26-CAG-FRT-mTFP1, in which the loxP-flanked stop cassette was excised in the germline, requires only Flp to activate mTFP1 expression. © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. 1. Introduction The most commonly used recombinase system in mouse genet- ics is Cre/LoxP, which is derived from bacteriophage P1. While less utilized, the Flp/Frt system, which is derived from Saccharomyces cerevisiae, has also proven to be a useful tool for mammalian genet- ics (Branda and Dymecki, 2004). Cre and Flp are both able to induce intra-molecular recombination between their recognition sites when oriented in the same direction in cis, including the deletion of the flanked sequence (Nagy, 2000). As Cre and Flp recognize different target sequences, the combined use of these recombi- nases would facilitate more sophisticated genetic manipulation of restricted cell populations. For example, intersectional genetic strategies using Cre and Flp have been reported (Awatramani et al., 2001; Farago et al., 2006; Sousa et al., 2009; Miyoshi and Fishell, 2006). In the Rosa26 knock-in intersectional reporter mice (e.g., R26::FLAP, RC::PFwe, and RCE:dual), dual recombinase responsive indicator alleles were designed, to express marker ∗ Corresponding author at: Institute for Virus Research, Kyoto University, Shogoin- Kawahara, Sakyo-ku, Kyoto 606-8507, Japan. Tel.: +81 75 751 4011; fax: +81 75 751 4807. E-mail address: iimayosh@virus.kyoto-u.ac.jp (I. Imayoshi). proteins only when Cre and Flp have been expressed in the same cell. As the majority of genes in the developing nervous system are expressed in multiple cell populations, these intersectional approaches provide important means to reduce complexity, such that smaller subpopulations of genetically defined cells can be targeted (Imayoshi et al., 2011). Here, we describe the production of a similar reporter allele that expresses mTFP1 (bright teal fluorescent protein) after Cre- and/or Flp-mediated recombination. mTFP1 is the improved vari- ant of the tetrameric CFP (cyan fluorescent protein) cFP484 from Clavularia coral. mTFP1 is one of the brightest and most photostable fluorescent proteins reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and has high maturation rate. mTFP1 is a true monomer and have lower toxicity to live cells (Ai et al., 2008). 2. Materials and methods 2.1. Targeting vector construction The R26-CAG-LF-mTFP1 knock-in vector (see Fig. 1), contains an SA (splice acceptor sequence)-puromycin resistance gene, a CAG promoter derived from pCAGGS (Niwa et al., 1991), a floxed 0168-0102/$ – see front matter © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved. doi:10.1016/j.neures.2012.02.003