Figure 1. Inversion – Excision strategy and genetic crosses for generating and validating Brn3c^Cre line.

a–d, Targeting strategy and recombination steps for the Brn3c locus. a, Brn3c^CKOCreNeo locus; b, Brn3c^CKOCre locus after removal of the Neo selection cassette by Flp - mediated recombination; c, Brn3c^Cre locus after Dre mediated inversion-excision at the rox12 – roxP sites - the Cre recombinase cDNA is now expressed at the original start site and the Brn3c gene is transcribed in reverse orientation (Chuang et al., 2016); d, Brn3c^WT, wild type configuration. Tick marks represent 1 kb intervals and SS marks intervals of indicated length. Coding regions and UTRs are indicated by green and white boxes and splice junction connections by angled black lines. Homologous recombination targeting arms (black lines) and southern blot probes are indicated under the locus. Diagnostic restriction enzymes : EcoRV (RV), Hind III (HIII). Genetic elements are as follows: triple repeat SV40 polyA (3xpA) functions as a bidirectional transcription stop = bidirectional red arrow; Cre recombinase - orange arrow; roxP (cyan triangle) and rox12 (white triangle) sites flank the endogenous gene; FRT sites (purple circles), flank the PGK-Neo Cassette (dark blue rectangle). Genotyping primers (thin black arrowheads, see also Supplementary table 1) are as follows: P1–P2 placed in the 5’ UTR, spanning the ATG in the wild type (211 bp) and ATG and 5’ FREX site (rox12 – roxP, 363 bp) in the targeted locus; P3–P4 = Cre recombinase (366 bp) ; P5–P6 = junction between the Cre recombinase and the Neo cassette (602 bp). Dre-mediated inversion excision can be tested using P1’–P5 (411 bp). Predicted southern blot fragment lengths are as follows: 5’ probe recognizes EcoRV fragments of 13142 bp for the wild type and 12558 for the targeted locus, and the 3’ probe recognizes HindIII fragments of 6969 bp for the wild type and 5546 for the targeted locus. e, Representative Southern blot for the 3’ probe on HindIII digested ES cell genomic DNA from two Brn3c^CKOCreNeo/WT (seen in a, lanes 2–3) and two Brn3c^WT/WT (seen in d, lanes 4–5) clones. MW – molecular weight markers of indicated length. Wild type band (6.7 kb) and mutant band (5.5 kb) are indicated. f, representative genotyping results from Brn3c^CKOCre/WT; Actin:FlpO mice and littermate controls. Molecular Weight (MW) markers are indicated on the left, and relevant PCR products (i–vi) on the right. Genotypes are Brn3c^CKOCre/WT; Actin:FlpO (mice iii, v and vi), Brn3c^CKOCreNEO/WT (mice i and ii), and Brn3c^WT/WT; Actin:FlpO (mouse iv). The targeted Brn3c allele is present in mice i, ii iii, v and vi, as seen by positive P3–P4 (Cre) and P1–P2 (FREX cassette, upper band, at 363 bp) reactions. The PGK Neo cassette is present only in mice i and ii (P5–P6 positive), which are negative for the FlpO reaction (P7–P8). The Neo cassette has been excised in mice iii, v and vi (P5–P6 negative), which are FlpO positive (P7–P8 positive). g, h, j Sequential Dre - Cre recombination. h, schematic of recombination cascade. Dre recombinase, transcribed from an ubiquitous promoter (CAG, h1) produces an inversion-excision between the rox12 - roxP sites, resulting in the inactivation of the Brn3c gene, and expression of the Cre cDNA (h2), which then can target the Rosa26^AP reporter (h3) (Tudor C Badea, Hua, et al., 2009). g, genotyping results for mice i–iv, the result of the breeding schematic shown in j. Genotype for mice i and ii is CAG:Dre; Rosa26^AP, genotype for mouse iii is CAG:Dre; Brn3c^CKOCre/WT; Rosa26^AP, and mouse iv is CAG:Dre; Brn3c^CKOCre/WT. The P1–P2 reaction is negative for all mice (no bands at 363 bp, only WT band at 211), however mice iii and iv, are positive for Cre (P3–P4) and inversion – excision (see c, P1–P5). Thus, in mice iii and iv the Cre has been inverted to adjoin the 5’ UTR (see panel c). Mice carrying the CAG:Dre; Brn3c^CKOCre/WT inversion-excision configuration were bread out to isolate the inverted Brn3c^CKOCre/WT locus, henceforth Brn3c^Cre/WT (panel c). i, k–m, Brn3c^Cre/WT mice crosses to three distinct reporters, (Brn3a^CKOAP/WT, k, Brn3b^CKOAP/WT , l, and Rosa26^tdTomato , m). For further details regarding the genetic strategy see also material and methods.