Binary recombinase systems for high-resolution conditional mutagenesis

Abstract

Conditional mutagenesis using Cre recombinase expressed from tissue specific promoters facilitates analyses of gene function and cell lineage tracing. Here, we describe two novel dual-promoter-driven conditional mutagenesis systems designed for greater accuracy and optimal efficiency of recombination. Co-Driver employs a recombinase cascade of Dre and Dre-respondent Cre, which processes loxP-flanked alleles only when both recombinases are expressed in a predetermined temporal sequence. This unique property makes Co-Driver ideal for sequential lineage tracing studies aimed at unraveling the relationships between cellular precursors and mature cell types. Co-InCre was designed for highly efficient intersectional conditional transgenesis. It relies on highly active trans-splicing inteins and promoters with simultaneous transcriptional activity to reconstitute Cre recombinase from two inactive precursor fragments. By generating native Cre, Co-InCre attains recombination rates that exceed all other binary SSR systems evaluated in this study. Both Co-Driver and Co-InCre significantly extend the utility of existing Cre-responsive alleles.

Figure 1.

Evaluation of Co-Driver candidate recombinases. (A) Diagrams of standardized SSR-driver and respective reporter plasmids that are co-transfected with a constitutively expressing mCherry plasmid into HEK293T cells. Transfected cells are mCherry⁺, and SSR activity is detected for Bxb1 using an inverted ZsGreen reporter or a STOP-cassette-based ZsGreen reporter for KD, B3 and Dre. Arrowheads indicate the orientation of individual SSR recognition sites. (B) Flow cytometric analysis of cells 24 h after transfection with Bxb1, KD, B3 or Dre recombinase and respective reporter constructs (top panels) or reporter constructs alone (bottom panels). Numbers within histograms represent the percentage of transfected cells showing recombination activity. Insets, scatter plots showing living cells and gating for mCherry⁺ cells (red rectangle). FI, relative fluorescence intensities reported in arbitrary units. (C) Quantification of recombination efficiency for Co-Driver candidates. Bars represent the percentage of cells showing recombination activity in subsets of mCherry⁺ cells [low, medium (med) or high mCherry expression; gating within the mCherry⁺ gate is shown right to bar graph]. Average data of three independent experiments with standard deviation and normalized to 100% Cre average recombination efficiency are shown. KD activity was not detectable (n.d.) in mCherry⁺ cells except in the mCherry^high population. Statistics: two-way ANOVA with Bonferroni post hoc test; n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2.

Dre-dependent activation of Cre. (A) Diagram depicting Dre-dependent Cre driver candidates 5′ 3× poly(A), 5′ CAT-poly(A) and Roxed-Cre with distinct rox-flanked STOP cassette configurations. In the case of Roxed-Cre, the Cre protein-coding sequence is interrupted by a rox-flanked STOP cassette inserted in between the codons of amino acids (aa) 59 and 60. (B) Activity of Cre driver candidates in transfected (mCherry⁺) HEK293T is detected using a ZsGreen reporter plasmid. Representative flow plots 24 h after transfection of HEK293T cells with a loxP-flanked ZsGreen reporter, Cre-Driver candidates and either omitting (top row) or adding (bottom row) Dre. (C) Quantification of recombination activity of Cre driver candidates with or without Dre Co-Driver. Bars represent the percentage of transfected cells showing recombination activity. Average data of three independent experiments are shown with standard deviation and normalized to 100% Cre average recombination efficiency. (D) MEFs derived from hemizygous Ai6 reporter mice (MEF-Ai6) express ZsGreen from the single copy Ai6 transgene (D top panel; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element) upon Cre recombination (D right panel; fluorescence microscopic image of MEF-Ai6 72 h after transfection with Cre). Combined, but not single transfections of Co-Driver modules Dre and Roxed-Cre result in excision of the loxP-flanked Ai6 STOP cassette, as detected by the presence of a 0.4-kb PCR fragment using primers located upstream and downstream of the STOP cassette (D bottom panel) and expression of ZsGreen, as detected by flow cytometry (E) in MEF-Ai6 72 h post-transfection.

Figure 3.

Recombination efficiency of Co-Driver and Co-InCre. (A) Sequential binary SSRs Co-Driver and Bxb1-rcCre comprise expression constructs for the primary recombinases Dre and Bxb1, which subsequently activate expression of functional Cre by removal of a rox-flanked STOP cassette or by inversion of attB/attP-flanked reverse complement (rc) Cre, respectively. Rectangles with arrows represent expression constructs and rounded rectangles represent proteins. (B) Coincidental binary SSRs generate active Cre from split-proteins (Cre-N, aa 19–59; Cre-C, aa 60–343; white rectangles) fused to protein reconstitution domains (blue rounded rectangles) by GCN4-coiled-coil (cc)-mediated dimerization [Split Cre (6)], DnaE-mediated protein trans-splicing yielding Cre with inserted non-native amino acids [split-intein-split-Cre, (9)] and Co-InCre reconstituting native Cre by gp41-1-mediated seamless protein trans-splicing. (C) MEF-Ai6 were transfected with a plasmid for constitutive mCherry expression and either two plasmids for the expression of both binary SSR modules or a single Cre plasmid. Representative flow plots 72 h post-transfection show the percentage of transfected cells with recombination activity for sequential binary SSRs and Cre (top) and coincidental binary SSRs (bottom). Transfected binary SSR systems are indicated on top of the flow plots. Insets show gating for transfected cells. (D) Recombination efficiency, defined as ratio of mCherry⁺ cells showing ZsGreen-expression, was determined in subsets of mCherry⁺ cells with low, medium (med) or high mCherry FI (gating within the mCherry⁺ gate is shown in the graph). Binary SSRs with sequential or coincidental mode of action are color-coded in shades of red, or blue, respectively. Average data of three independent experiments are shown with standard deviation and normalized to 100% Cre recombination efficiency. Statistics: two-way ANOVA with Bonferroni post hoc test; n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4.

Co-Driver and Co-InCre trigger efficient recombination in the developing mouse brain. (A) A constitutively expressing EGFP plasmid and plasmids encoding either the Co-Driver or the Co-InCre components were electroporated into the developing brains of embryonic day 14.5 (E14.5) hemizygous Ai14 reporter mice. Upon Cre recombination, these mice express tdTomato from a single reporter gene integrated into the ROSA26 locus (WPRE). (B) Coronal sections of fluorescence-positive brain areas (white dashed line) were prepared from post-natal Day 9–10 (P9–P10) mice. The post-natal brain of an animal electroporated with EGFP and Cre plasmids is shown. (C–F) Maximum intensity projections (MIP) of electroporation-positive brain areas (coronal brain sections). (C) Cre-induced tdTomato expression can only be detected by confocal imaging when both Co-Driver components, Dre and Roxed-Cre, were electroporated. Fluorescent signals are combined with phase-contrast images of brain sections. (D) High magnification MIP of recombination-positive areas in the cortex (left) and long-range axonal projections in the corpus (c.) callosum (right). (E) Both Co-InCre N- and C-terminal fragment evoke recombination in electroporation-positive areas, while single components show no recombinase activity. (F) Control electroporation using full-length Cre recombinase. Scale bar for panels (C), (E) and (F), 500 µm. Scale bars for panel (D), 20 µm.

Figure 5.

Sequential expression of Co-Driver components during development of the mouse neocortex. (A) Co-Driver components Dre [fused to the porcine teschovirus-1 (P)2A sequence and blue fluorescent protein, BFP] and Roxed-Cre were introduced into a human (h)GFAP promoter expression vector and the Thy1.2 expression vector. E14.5 embryos carrying a single-copy Cre-inducible tdTomato reporter gene (Ai14) were electroporated with a constitutively expressing EGFP plasmid and either hGFAP-Dre with Thy1.2-Roxed-Cre (white rectangles) or Thy1.2-Dre with hGFAP-Roxed-Cre (gray rectangles). (B) Representative single-plane confocal images of fluorescence-positive areas within the cortex and MIP of areas in proximity to the corpus (c.) callosum of post-natal day (P)10 mice are shown (top panel, hGFAP-Dre with Thy1.2-Roxed-Cre electroporations; bottom panel, Thy1.2-Dre with hGFAP-Roxed-Cre electroporations). Arrow heads denote cells within the cortex that show both EGFP and tdTomato fluorescence, arrows denote cells with single tdTomato fluorescence. Scale bars, 100 µm. (C) Quantification of EGFP-positive cells (left), tdTomato-positive cells (middle) and tdTomato/EGFP double-positive cells (right) in coronal brain sections. Average data of three animals and three coronal sections per animal with standard deviation are shown. Statistics: unpaired Student’s t-test; ***P < 0.001.

Figure 6.

Binary Co-Driver and Co-InCre recombinase systems for sequential and coincidental conditional transgenesis. The binary recombinase components are expressed using two promoters with distinct expression profiles (red and cyan arrows, top right). Hypothetical temporal sequences of promoter activities within two distinct developmental stages of a cell lineage are shown on the left. Once activated, Cre recombinase processes a generic loxP-flanked responder sequence (dark gray box, processing is indicated by a linear and a circular reaction product), which could either represent a floxed exon leading to gene ablation or a floxed STOP cassette resulting in expression of a transgene. Non-overlapping expression patterns (A, B) result in recombination of loxP sites by Co-Driver only when Dre is expressed first (A). Both Co-Driver and Co-InCre yield recombination of loxP sites in the case of coincidental promoter activation with similar (C) or different (D, E) durations of transcription from the individual promoters.