locus (R26CreER; 7,9,10). In the absence of 4HT, R26CreER;Z/AP mice typically exhibit no AP+ cells per retina, whereas R26CreER;Brn3aCKOAP/+ and R26CreER;Brn3bCKOAP/+ mice exhibit several hundred AP+ RGCs per retina. A similar differential in the number of AP+ neurons is observed following in utero or early postnatal exposure to 4HT, with a single IP injection of 100– 200 mg in the early postnatal period producing several dozen AP+ RGCs per R26CreER;Z/AP retina and many thousands of AP+ RGCs per R26CreER;Brn3aCKOAP/+ or R26CreER;Brn3aCKOAP/+ retina [7,10]. For sparse labeling of neurons without selection for a particular subtype, the R26CreER;Z/AP combination with an early postnatal injection of 4HT generates a frequency of labeling that is close to optimal for morphologic analyses [(0.001%–0.01% of neurons labeled; 5,10]. When the Z/AP reporter is used in conjunction with a set of cell-type selective CreER lines – produced by knocking an IRES-CreER into the 39 untranslated region (UTR) of the genes coding for choline acetyl transferase (ChAT-IRES-CreER) or tyrosine hydroxylase (TH-IRES-CreER) - the low level of CreER production, together with the relatively small number of catecholaminergic or cholinergic neurons, results in extremely sparse cell labeling [4]. In the present work we have enhanced the sparse labeling technique by developing two Rosa26 knock-in mouse lines: one line increases the dynamic range of CreER action, and the second line exhibits Cre-mediated activation of AP with high efficiency and with undetectable background AP activity in unrecombined cells. The R26rtTACreER line uses the reverse tetracycline transactivator (rtTA) and doxycycline to control the expression of CreER, with the result that 4HT-induced Cre recombinase activity can be set at a level that is substantially lower than that typically obtained with constitutively expressed CreER lines. Thus the R26rtTACreER line permits precisely timed sparse recombination of target loci with a high efficiency of Cre-mediated recombination. Because the Rosa26 locus is widely expressed, the R26rtTACreER line can be used to generate sparse genetic mosaics to test the behavior of individual mutant cells in a wide variety of tissues and/or in situations in which the conventional allele is lethal. The R26IAP line complements the popular but inefficiently recombin- ing Z/AP line by providing an AP reporter that exhibits high efficiency Cre-mediated recombination in virtually any cell. The R26IAP line should prove especially useful when paired with CreER loci that exhibit low levels of 4HT-induced Cre activity. We demonstrate the utility of the latter line by using it to: (a) assess the efficiency of Cre-mediated recombination following either direct uptake of Cre protein or infection by an adenovirus vector expressing Cre, (b) generate brain atlases of sparsely labeled cholinergic and catecholaminergic neurons, and (c) characterize the morphologies of type 1 dopaminergic retinal amacrine cells, which have axon-like arbors that are among the largest of any cells in the mammalian retina. Results R26rtTACreER: Dual Control of Cre Action by Doxycycline and 4-Hydroxytamoxifen (4HT) To construct a ubiquitously expressed CreER(T) allele (hereafter referred to as ‘CreER’; 2) with a large dynamic range of CreER expression, we generated the Rosa26 knock-in allele R26rtTACreER (Figure 1A,B). In this allele, CreER is under the control of the reverse tetracycline trans-activator (rtTA), a sequence-specific DNA-binding protein that activates transcription upon binding tetracycline derivatives such as doxycycline [11]. The rtTA coding sequences are transcribed as part of the ubiquitously expressed New Cre/lox Genetic Tools Figure 1. Structures of the R26rtTACreER and R26IAP knock-in alleles. (A) The WT Rosa26 locus showing the location of the ubiquitous transcript derived from this locus and the unique Xba I site into which the rtTACreER and IAP cassettes were inserted. (B) The R26rtTACreER locus. The rtTA coding region is preceded by a splice acceptor so that it can be spliced onto the 59 exon of the endogenous Rosa26 transcript, and it is followed by multiple transcription termination signals (‘‘stop’’). In the presence of doxycycline, the rtTA protein activates transcription from the seven head-to-tail Tet response elements immediately 59 of a minimal cytomegalovirus immediate early promoter (‘‘TRE’’), leading to expression of the CreER coding region. The resulting CreER protein translocates to the nucleus upon binding to 4- hydroxytamoxifen (4HT). (C) The R26IAP locus in its germline configuration (upper diagram) and following Cre-mediated recombina- tion (lower diagram). The IAP region is preceded by a splice acceptor so that it can be spliced onto the 59 exon of the endogenous Rosa26 transcript. In the germline configuration, the two loxP sites in head-to- head orientation surround a central region that must be inverted to generate a correctly configured human placental alkaline phosphatase (hPLAP) minigene (lower diagram). In the orientation shown in the lower diagram, one loxP site resides in the intron and the second loxP site resides in the 39 UTR. For the R26rtTACreER allele, the frt-flanked PGK-Neo selectable marker used during ES culture was removed by Flp- mediated recombination in the mouse germline. Scale bars in A, B and C are 1 Kb. doi:10.1371/journal.pone.0007859.g001 Rosa26 transcription unit and are joined by splicing to the 59 exon of this transcript. Upon administration of doxycycline (in food pellets and/or drinking water), rtTA activates transcription of the CreER coding region by binding to seven tandem copies of the Tet operator located immediately 59 of a minimal promoter. To systematically explore the relationship between the level and timing of doxycycline and 4HT exposure and the efficiency of Cre- mediated recombination, we analyzed the expression of AP in RGCs using R26rtTACreER;Brn3aCKOAP/+ and R26rtTACreER;Brn3bCKOAP/+ mice (Figure 2; see Figure 1 of [reference 7] for a schematic of these reporters). Doxycycline was delivered in food pellets at 1.75 mg/g and/or in drinking water at 0.5, 1, or 2 mg/ml. 4HT dissolved in vegetable oil was delivered to pregnant females or to early postnatal mice as a single IP injection. Retina flat mounts were prepared from adult mice, stained histochemically for AP activity, and scored as PLoS ONE | www.plosone.org 2 November 2009 | Volume 4 | Issue 11 | e7859