Mouse strain generation

All animal procedures were performed in accordance with the National Institutes of Health Guide for the care and use of laboratory animals and approved by local institutional animal welfare committees (associated with Baylor College of Medicine, University of Tübingen, or University of Oslo). The tTA image repository holds image data from 12 adult bigenic mice, representing intercrosses between five promoter-tTA driver lines and two tetO-lacZ reporter lines (Table 1). Experimental details about animal constructs are provided below. Microscopic images from Prnp, Camk2a, and Nop driver lines^4,21,22 were imported from the Rodent Brain Workbench (www.rbwb.org). Data from single transgenic (tetO-lacZ) control animals, excluding the possibility of endogenous β-galactosidase staining, have been reported earlier²². Information on the three strains used to generate Pcp2 and Pitx3 lacZ expression data is provided below.

The lacZ reporter line used for detection of tTA activity in the Pcp2, Nop, and Pitx3 driver lines encoded an in-frame fusion of the E. coli lacZ gene with green fluorescent protein (GFP) under control of the first-generation tetO promoter²⁵. The reporter construct also included the nuclear localization signal (nls) from the simian virus 40 (SV40) large T antigen and was followed by the three prime untranslated region (3′ UTR) from mouse calcium-calmodulin kinase type IIα (Camk2a). It is unclear from the original report whether the tetO-lacZ-nls-GFP animals were generated on a B6CBA F2 or B6SJL F2 background, but in either case the line was eventually maintained by backcross with C57BL/6 (Dr. Mark Mayford, personal communication). This line was obtained from Dr. Mark Mayford (Department of Neuroscience, Scripps Research Institute, La Jolla, CA, USA) in 2010 and backcrossed to C57BL/6 J for another 4 generations before being mated with the Pcp2-tTA and Pitx3-tTA driver lines as described below.

Mice expressing tTA under control of the mouse L7/Purkinje-cell protein 2 (Pcp2) promoter were created by Dr. Harry T. Orr (Department of Laboratory Medicine and Pathology, University of Minnesota, USA) and obtained from a colony derived from this stock by Dr. David Nelson (Department of Molecular and Human Genetics, Baylor College of Medicine, USA). The original line was created by injection into FVB/N embryos and maintained on an FVB background. It was received in 2011 and backcrossed to FVB/NJ for another 5 generations before outcrossing to the tetO-lacZ-nls-GFP reporter (see above) for the current studies. The line is available through Jackson Laboratories under strain name FVB-Tg(Pcp2-tTA)3Horr/J, stock #5625.

Pitx3-tTA mice were generated in the laboratory of Dr. Huaibin Cai (Laboratory of Neurogenetics, National Institute of Aging, Bethesda, MD, USA) by targeted insertion of an IRES2-tTA-loxP-neomycin-loxP cassette in exon 4 of the mouse Pitx3 gene¹⁹. 129/SvJ ES founder males were generated and their offspring were mated with a Cre line to remove the neomycin cassette from the transgene. The line was backcrossed to produce congenic C57BL/6J animals. Animals were obtained from Dr. Mark Mattson (Laboratory of Neurosciences, National Institute of Aging, Baltimore, MD, USA) in 2013 that had been derived from Dr. Huaibin Cai’s original colony at the NIH. The mice were bred at Baylor College of Medicine, Houston, TX, USA for one generation with C57BL/6J before outcrossing the offspring with the tetO-lacZ-nls-GFP reporter line for the current study.

Description of the construction of Prnp, Camk2a, and Nop-tTA lines are provided in previous publications^4,21,22. In brief, Prnp-tTA/tetO-lacZ and Camk2a-tTA/tetO-lacZ mice were generated by crossing a Prnp promoter line (Prnp-tTA, line F959²⁵) or Camk2a promoter line²⁶ with a responder line transgenic for a bidirectional reporter gene construct containing the E. coli derived LacZ reporter gene encoding β-galactosidase^27,28, and the Luciferase gene from Photinus pyralis (not used in the present study) (Luc-tetO-lacZ). Nop-tTA mice were generated by backcrossing the Nop-tTA mouse line provided by Dr. Mark Mayford (Line tTA-EC²⁹; also known as Klk8-tTA; MMRRC strain # 031780) to C57BL/6 J for 1–3 generations before outcrossing to the responder E. coli lacZ and green fluorescent protein (GFP) responder line listed above (tetO-lacZ-nls-GFP)²⁶.