Figure 1

TEMTAC system components.

(a) Cre-LoxP mediated generation of plasmid fusions is shown in a schematic view. Acceptor A plasmid module is incubated with two Donor modules, D1 and D2 in the presence of Cre recombinase. Concomitant assembly (Cre) and excision (De-Cre) reactions occur until equilibrium is reached. Acceptor-Donor (A-D1, A-D2) and Acceptor-Donor-Donor (A-D1-D2 or “pTEMTAC”) fusion plasmids co-exist with educt plasmids when equilibrium is reached. Acceptor A contains a common origin or replication (ColE1), Donors D1 and D2 contain conditional origins of replication derived from phage R6Kγ, rendering their propagation in regular cloning strains dependent on productive Cre fusion with Acceptor A. (b) Donor D1 (pMDC-RNAiDual) is shown in a schematic view. This Donor provides cassettes for multiple shRNA production. (c) shRNA-mediated downregulation of SHP2, HRAS and BRAF after transfection with a Donor D1 producing specific shRNAs. Transfected HEK293 (for HRAS and BRAF) or GH-HEK293 cells were lysed and analysed by Western blotting. (d) Four Donor plasmid variants D2.1 to D2.4 are shown schematically, which realize four distinct dynamic ranges of exogenous protein expression. Abbreviations: Cre, Cre recombinase enzyme; LoxP, imperfect inverted repeat recognized by Cre; GOI, gene of interest; A-D1, fusion of Acceptor A with Donor D1; A-D2, fusion of Acceptor A with Donor D2 (or variants); A-D1-D2, complete fusion of Acceptor A with Donors D1 and D2 (or variants); shRNA, small hairpin RNA sequence; I-SceI, PI-PspI and PI-SceI are homing endonucleases; H1, U6, CMV and CAG are common mammalian active promoters; pA and SV40 are common poly-adenylation signals; TRE-pCMVmin, tetracycline response element with minimal CMV promotor; rtTA, tetracycline transactivator with (random) mutagenesis derive Tet repressor part of the transactivator gene; Sp, Cm, Hygr and Zeo denote resistance marker genes for spectinomycin, chloramphenicol, hygromycin and zeocin, respectively; YFP, yellow fluorescence protein; TetR, tet repressor gene; TetR-KRAB, tetracycline-controlled hybrid protein of TetR with the KRAB silencing domain of human Kid1; IRES, internal ribosome entry site; Frt, FLP recognition target; ColE1, common colicin E1 derived replication origin; R6Kγ, conditional origin derived from R6Kγ phage.