Supplementary Figure 10: Generation of Crhr1-ires-Cre mice.

Strategy for targeted manipulation of the Crhr1 locus. (a) Wild-type Crhr1 locus depicting the position of integration of the docking site for recombinase-mediated cassette exchange (RMCE) in Intron 2. For better visualization different scales were used for exons and introns to illustrate the Crhr1 gene structure. (b) Crhr1^tZ-ΔloxP allele containing the RMCE docking site in intron 2. A magnification of the docking site is provided above the Crhr1^tZ-ΔloxP allele. The targeting vector for RMCE equipped with respective attB sites is depicted below. Of note, loxP, frt, attB, attP, and T2A sites are not to scale. (c) ΦC31-mediated cassette exchange in embryonic stem (ES) cells resulted in integration of the targeting vector into the right attP site. ES cells were used to generate mice, which were subsequently bred to FLPeR mice to simultaneously remove the reporter and selection cassettes. (c) The correct integration and removal of selection and reporter cassettes was verified by PCR. (d) Detection of Flp recombinase-mediated removal of Tau-lacZ reporter cassette with PCR. (e) Confirmation of simultaneous Flp recombinase-mediated removal of the hygromycin selection cassette. (f) Detection of Cre recombinase expression cassette in Crhr1 locus. PCRs in d-f were independently replicated in each individual animal (within the respective mouse line) that was genotyped (n > 20).