New Mouse Lines for the Analysis of Neuronal Morphology Using CreER(T)/loxP-Directed Sparse Labeling Tudor C. Badea1,4*, Zhong L. Hua1, Philip M. Smallwood1,4, John Williams1,4, Thomas Rotolo1,4, Xin Ye1, Jeremy Nathans1,2,3,4* 1 Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 2 Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 3 Department of Ophthalmology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America, 4 Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America Abstract Background: Pharmacologic control of Cre-mediated recombination using tamoxifen-dependent activation of a Cre- estrogen receptor ligand binding domain fusion protein [CreER(T)] is widely used to modify and/or visualize cells in the mouse. Methods and Findings: We describe here two new mouse lines, constructed by gene targeting to the Rosa26 locus to facilitate Cre-mediated cell modification. These lines should prove particularly useful in the context of sparse labeling experiments. The R26rtTACreER line provides ubiquitous expression of CreER under transcriptional control by the tetracycline reverse transactivator (rtTA); dual control by doxycycline and tamoxifen provides an extended dynamic range of Cre-mediated recombination activity. The R26IAP line provides high efficiency Cre-mediated activation of human placental alkaline phosphatase (hPLAP), complementing the widely used, but low efficiency, Z/AP line. By crossing with mouse lines that direct cell-type specific CreER expression, the R26IAP line has been used to produce atlases of labeled cholinergic and catecholaminergic neurons in the mouse brain. The R26IAP line has also been used to visualize the full morphologies of retinal dopaminergic amacrine cells, among the largest neurons in the mammalian retina. Conclusions: The two new mouse lines described here expand the repertoire of genetically engineered mice available for controlled in vivo recombination and cell labeling using the Cre-lox system. Citation: Badea TC, Hua ZL, Smallwood PM, Williams J, Rotolo T, et al. (2009) New Mouse Lines for the Analysis of Neuronal Morphology Using CreER(T)/loxP- Directed Sparse Labeling. PLoS ONE 4(11): e7859. doi:10.1371/journal.pone.0007859 Editor: Tsuneya Ikezu, University of Nebraska Medical Center, United States of America Received August 12, 2009; Accepted October 20, 2009; Published November 16, 2009 Copyright: 2009 Badea et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research has been funded by the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: tbadea@mail.jhmi.edu (TCB); jnathans@jhmi.edu (JN) Introduction Altering the mouse genome by Cre-mediated recombination is one of the central technologies of modern mammalian genetics [1]. An important adjunct to this technology has been the development of a method for pharmacologic control of Cre- mediated recombination by fusing Cre with a mutant estrogen receptor ligand-binding domain that recognizes 4-hydroxytamox- ifen (4HT) rather than estrogen [CreER(T); referred to hereafter as CreER; 2,3]. The CreER protein is sequestered in the cytosol until 4HT exposure releases it for nuclear migration. For typical applications of the CreER technology - for example, experiments involving timed inactivation of conditional alleles - highly efficient recombination is considered desirable. By contrast, relatively inefficient recombination is essential for experiments in which the goal is to monitor the behavior, morphology, or function of individual genetically modified cells in an environment populated by unmodified neighbors. In extreme cases - for example, experiments using genetically encoded reporters to study the morphologies of very large CNS neurons - the efficiency of recombination must be lowered to fewer than 20 cells per mouse [4]. Other applications have used sparse labeling with Cre/loxP recombination to study neuronal morphologies in the retina [5], to define the role of the Frizzled5 receptor in the survival of thalamic neurons [6], and to compare the roles of transcription factors Brn3a and Brn3b in retinal ganglion cell (RGC) development [7]. One limitation of the mouse lines currently available for sparse labeling relates to the efficiency of Cre-mediated recombination, which varies substantially with different combinations of CreER source and loxP target. For example, we observed that the Z/AP line, which expresses human placental alkaline phosphatase (AP) upon excision of a loxP-flanked beta-geo/transcriptional stop cassette [8], recombines with an efficiency ,1,000 lower than similarly designed conditional AP knock-in alleles of the Brn3a and Brn3b genes (Brn3aCKOAP and Brn3bCKOAP), when each target gene is tested with a ubiquitously expressed CreER knock-in at the Rosa26 PLoS ONE | www.plosone.org 1 November 2009 | Volume 4 | Issue 11 | e7859