precisely controls the production of one or more molecules that are limiting for process formation. Methods ES Cells and Mouse Husbandry Knock-in constructs were designed with inserts at the Xba I site at the Rosa26 locus as indicated in Figure 1A. ES cells were electroporated and colonies were selected in G418 and screened by Southern blot hybridization with the 59 flanking probe shown in Figure 1A. Karyotypically normal ES cells were used for blastocyst injection. Following germline transmission of the targeted allele, the PGK-neo cassette of R26rtTACreER was excised by crossing to mice expressing Flp in the germline. The R26rtTACreER and R26IAP lines have been maintained as homozygous stocks on a mixed Sv1296C57BL/6 background. Both lines have been deposited at the Jackson Laboratories and are freely available. Mice were handled and housed in accordance with the Johns Hopkins University Animal Care and Use Protocols and IACUC guidelines. Genotyping PCR primers for genotyping are as follows. R26IAP [sense strand, located in the first exon of AP: GGCAACGAGGT- CATCTCCGTGATGAA; antisense strand, located in the intron in the inverted configuration: GGTTGCCTGGGTCTGAGC- TAGTGCC; product size: 290 bp); R26rtTATetCreER [sense strand: CCCTCGTGATCTGCAACTCCAGTC ( = TB29); an- tisense strand: TAGAATCGGTGGTAGGTGTC; product size: 700 bp]; WT [(i.e. the untargeted) Rosa26 locus [sense strand: TB29, listed above; antisense strand: GGAGCGGGAGAAATG- GATATG; product size: 500 bp]. PCR was performed with Advantage 2 Polymerase (Clontech, La Jolla, CA) using 35 cycles of 30 seconds denaturation at 94uC, 30 second annealing at 60uC, and 1 minute elongation at 72uC. His-NLS-Cre and Ad-Cre An E. coli expression plasmid encoding His-NLS-Cre [13] was a kind gift of Dr. H. Earl Ruley (Vanderbilt University). His-NLS- Cre expressed in E coli was purified to .90% homogeneity by nickel chelate affinity chromatography. The protein was eluted in 500 mM imidazole, and concentrated and desalted by ultrafiltra- tion against PBS supplemented with 150 mM NaCl. For IP injection of His-NLS-Cre, 100 ml of a 1.84 mg/ml stock was injected per adult mouse; for intraocular injection 2 ml of a 3.52 mg/ml stock was injected per eye at P5 using a 30 gauge needle. For IV injection into the tail vein, 400 ml of an Ad-Cre stock (36109 plaque forming units/ml; Virenz, Baltimore, MD) was injected per adult mouse; for intraocular injection, 1.5 ml of the same virus stock was injected per eye at .P21 with a glass micro-needle. Tissues were analyzed .14 days later for IP His- NLS-Cre and IV Ad-Cre injections, and .10 days later for intraocular injections. Doxycycline and 4HT Delivery Doxycycline (Sigma, St. Louis, MO) was mixed with drinking water and/or delivered as premixed food pellets (Research Diets, Inc., New Brunswick, NJ). For experiments with R26rtTACreER References New Cre/lox Genetic Tools mice, 4HT (98% pure) was dissolved in ethanol, mixed with sunflower seed oil (Sigma, St. Louis, MO), centrifuged under vacuum to remove the ethanol, and delivered as a single IP injection. For experiments with R26IAP mice, the same protocol was followed except that 70% purity 4HT (Sigma) was used for some experiments. For R26IAP mice, IP injections of 4HT were performed between P3 and P8 at a dose of 0.03–0.04 mg/g body weight. Tissue Processing Tissue processing and AP staining were performed essentially as described in ref. 10. For tissues other than eyes, mice were anesthetized and subjected to transcardiac perfusion with 4% paraformaldehyde in PBS. The brain, liver, or kidney was set in a block of 3% low-melting point agarose in PBS and a series of 300 mm sections were cut with a vibratome. Segments of abdominal wall and stomach were processed intact. Retinas were dissected from eyes that had been immersion fixed in 4% paraformaldehyde in PBS, and were gently flattened with a plastic mesh. Tissues in PBS were heated in a water bath at 69uC for 90 minutes to inactivate endogenous AP and, for those embedded in low melting point agarose, to melt the agarose surrounding the tissue; heat-inactivated tissues were then reacted in NBT/BCIP for several hours to overnight at room temperature with gentle agitation. After washing and post-fixation, tissues were dehydrated with a graded ethanol series, and clarified in 2:1 benzylbenzoa- te:benzyl alcohol (BBBA). For long-term storage, samples were returned to ethanol. Microscopy and Neurite Tracing Bright-field images were captured on a Zeiss dissecting microscope equipped with OpenLab software, and bright-field and DIC images were captured on a Zeiss Axio Imager Z1 microscope equipped with a motorized stage and AxioVision software. For retina flat mounts, a montage of 5X images was obtained with the Zeiss Axio Imager Z1 microscope using an X-Y stage and the mosaics module and assembled using AxioVision software. For type 1 DA cells, in which dendrites and axon-like processes are confined to a single sublamina of the IPL, the AP- labeled neurites were traced in the X–Y plane without loss of information using Neuromantic software (www.rdg.ac.uk/neuro- mantic/). For coronal brain sections, Bregma positions and neuroanatomic structures were assigned as indicated in ref. 15. Acknowledgments The authors thank Se-Jin Lee and Suzanne Sebald for advice on the Tet system and materials for of the rtTA vector; D.J. Pan for the Ad-Cre virus; Earl Ruley for the kind gift of the His-NLS-Cre plasmid; Chip Hawkins, Ann Lawler, Johnisha Witherspoon, and Elizabeth Alexander for injecting ES cells into blastocysts; and Hao Chang for helpful comments on the manuscript. Author Contributions Conceived and designed the experiments: TCB JN. Performed the experiments: TCB ZLH PMS JW TR XY JN. Analyzed the data: TCB JN. Contributed reagents/materials/analysis tools: TCB ZLH. Wrote the paper: TCB JN. 1. Branda CS, Dymecki SM (2004) Talking about a revolution: the impact of site-specific recombinases on genetic analysis in mice. Dev Cell 6: 7–28. 2. Feil R, Brocard J, Mascrez B, LeMeur M, Metzger D, et al. (1996) Ligand- activated site-specific recombination in mice. Proc Natl Acad Sci USA 93: 10887–10890. PLoS ONE | www.plosone.org 11 November 2009 | Volume 4 | Issue 11 | e7859