New Cre/lox Genetic Tools Figure 2. Dose and timing of doxycycline and 4HT exposure on Cre-mediated recombination efficiency using R26rtTACreER. (A) Individual experiments are numbered, the doxycycline and 4HT regimens are shown schematically, and the number of mice with high/medium, sparse, or no AP+ labeling of RGCs is shown in the color-coded histograms at right. Experiments 1–21 and 22–33 were performed with R26rtTACreER;Brn3aCKOAP/+ and R26rtTACreER;Brn3bCKOAP/+ mice, respectively. (B–F) Representative retina flat mounts (B,C,E,F) and a 200 mm coronal brain section at the level of the superior colliculus (D) were obtained from the numbered experiments indicated in the lower right corner of each panel. A single IP injection was used to deliver 4HT either to the mother for in utero exposure or to individual pups for postnatal exposure. doi:10.1371/journal.pone.0007859.g002 having high/medium labeling (.50 AP+ RGCs per retina; Figure 2E, F), sparse labeling (1–50 RGCs per retina; Figure 2C), or no labeling (Figure 2B). Various dosing regimens were tested, of which 33 are shown diagrammatically in Figure 2A. For each of the two genotypes tested, the diagrams have been ordered with respect to recombination efficiency, presented in the histograms on the right side of Figure 2A. From these experiments several patterns emerge. First, initiating doxycycline exposure after approximately embryonic day (E) 5 gives low or no recombination, even if a large dose of 4HT is PLoS ONE | www.plosone.org 3 November 2009 | Volume 4 | Issue 11 | e7859