Gt(ROSA)26Sor^{em1.2(CAG-Rpl22,-Uprt,-mKate2,-Ago2)Jaws}

The Tagger transgene comprises an HA-tagged ribosomal protein L22 (Rpl22-HA, for Ribo-Tag), Uracil phosphoribosyltransferase from T.gondii (TgUPRT, for TU-Tag), mKate2 red fluorescent protein with a triple nuclear localization signal (mKate2, for Nuc-Tag), and FLAG-V5-tagged Argonaute2 (FLAG-V5-Ago2, for Ago-Tag). This cassette was placed under the control of the CAG promoter and two transcription-terminating STOP cassettes Cre-dependent (LoxP-STOP-LoxP, LSL) and Flp-dependent (Frt-NeoR-Frt, FNF). In the Tagger transgene, protein-coding cistrons were separated by a sequence encoding the Porcine Teschovirus 2A peptide (P2A) and flexible linkers (GSGSG) for improved separation. The transgene was targeted to the Rosa26 locus, including a ubiquitous CAG driver followed by Flp- (FNF, Frt-NeoR-Frt) and Cre-dependent (LSL, LoxP-STOP-LoxP) terminators (STOP cassettes). The inclusion of two terminators enables transgene activation at the intersection of cell populations expressing Cre and Flp driven by distinct promoters, to precisely target cells based on combinations of cell identity markers or activity. Tagger expression cassette (in R26LK-TK-Tagger vector) was targeted into Rosa26 safe harbor locus in V6.5 ES cells using CRISPR/Cas9 as described previously. In brief, 40 g of HTV linearized with AscI was co-electroporated with 5 g CRISPR/Cas9 nickase plasmid pX335 (Feng Zhang, Addgene plasmid #42335) encoding sgRNA-targeting the homology junction. Flp-mediated recombination removed the FRT-flanked neomyocin resistance cassette. Cre-mediated recombination removed the floxed STOP cassette. (J:279059)