Atlas of transgenic Tet-Off Ca²⁺/calmodulin-dependent protein kinase II and prion protein promoter activity in the mouse brain

Abstract

Conditional transgenic mouse models are important tools for investigations of neurodegenerative diseases and evaluation of potential therapeutic interventions. A popular conditional transgenic system is the binary tetracycline-responsive gene (Tet-Off) system, in which the expression of the gene of interest depends on a tetracycline-regulatable transactivator (tTA) under the control of a specific promoter construct. The most frequently used Tet-Off promoter mouse lines are the Ca²⁺/calmodulin-dependent protein kinase II (CamKII) and prion protein (PrP) promoter lines, respectively. To target the regulated gene of interest to relevant brain regions, a priori knowledge about the spatial distribution of the regulated gene expression in the brain is important. Such distribution patterns can be investigated using double transgenic mice in which the promoter construct regulates a LacZ reporter gene encoding the marker β-galactosidase which can be histologically detected using its substrate X-gal. We have previously published an atlas showing the brain-wide expression mediated by the Tet-Off PrP promoter mouse line, but the distribution of activity in the Tet-Off CamKII promoter mouse line is less well known. To compare promoter activity distributions in these two Tet-Off mouse lines, we have developed an online digital atlas tailored for side-by-side comparison of histological section images. The atlas provides a comprehensive list of brain regions containing X-gal labeling and an interactive dual image viewer tool for panning and zooming of corresponding section images. Comparison of spatial expression patterns between the two lines show considerable regional and cellular differences, relevant in context of generation and analysis of inducible models based on these two tetracycline responsive promoter mouse lines.

Introduction

Transgenic animal models allowing conditional regulation of gene expression are powerful tools for experimental investigation of neurodegenerative diseases (Lewandoski, 2001). A frequently used binary transgenic system is the tetracycline responsive gene (Tet-Off) system, in which transgene expression is activated or silenced by administration of tetracycline, or its derivatives (Gossen et al., 1995, Gossen and Bujard, 1992, Lewandoski, 2001). For brain-specific expression, conditional models using the Tet-Off system are currently mainly based on two transgenic mouse lines, either the prion protein (PrP)- or the Ca²⁺/calmodulin-dependent protein kinase II (CamKII) promoter mouse line. The PrP promoter line has been successfully used in several studies of various diseases, including prion diseases (Safar et al., 2005, Tremblay et al., 1998), Parkinson's disease (Nuber et al., 2008), analysis of alcohol consumption (Choi et al., 2002), spinocerebellar ataxia type 3 (Boy et al., 2009), and neurofilament transport (Millecamps et al., 2007). The CamKII promoter line has been used for the investigation of synaptic plasticity, learning and memory consolidation (Bukalo et al., 2004, Limback-Stokin et al., 2004, Mayford et al., 1997, Wood et al., 2005), neurodevelopmental disorders (Alvarez-Saavedra et al., 2007, Jerecic et al., 2001), and for the generation of transgenic mouse models for Alzheimer's disease (Jankowsky et al., 2005), Huntington's disease (Yamamoto et al., 2000), Parkinson's disease (Nuber et al., 2008), neocortical degeneration (Muyllaert et al., 2008), schizophrenia (Pletnikov et al., 2008), and neurofibromatosis (Saito et al., 2007).

Since the expression of the responder gene is dictated by the activity of the promoter construct, knowledge about the anatomical distribution of promoter activity is critical both for the successful generation of an inducible disease model, as well as for the interpretation of observations made in conditional models based on the PrP or CamKII promoters (Chen et al., 1998, Furth et al., 1994, Yamamoto et al., 2000). A priori insight in these anatomical distributions is therefore valuable for researchers constructing or using inducible Tet-Off models. CamKII is known as a forebrain specific promoter while the PrP promoter activity is mainly distributed in the brainstem and cerebellum (Bailly et al., 2004, Boy et al., 2006, Bukalo et al., 2004, Chen et al., 1998, Ghavami et al., 1999, Liang et al., 1996, Liu et al., 2001, Mantamadiotis et al., 2002, Mayford et al., 1995, Mayford et al., 1996, Tremblay et al., 1998, Alvarez-Saavedra et al., 2007, Yamamoto et al., 2000). At the cellular level, CamKII promoter activity is neuron specific and has not been reported in glia (Bukalo et al., 2004, Chen et al., 1998, Mayford et al., 1996, Mayford et al., 1997, Michalon et al., 2005, Alvarez-Saavedra et al., 2007), whereas PrP promoter activity is seen in both neuronal and glia cell populations (Boy et al., 2006, Boy et al., 2009, Gotz et al., 2001). Using double-transgenic mice with the LacZ reporter gene (encoding the enzymatically detectable β-galactosidase) as a responder, we have performed comprehensive brain-wide mapping of promoter activity at the level of regions and cells in PrP/LacZ (Boy et al., 2006) and CamKII/LacZ mice (present study).

We have previously published a detailed online atlas of the distribution of PrP promoter regulated gene expression (Boy et al., 2006). Building upon this technology, we here present a similar atlas of CamKII promoter-regulated gene expression distributions across the entire mouse brain. We further present a novel atlas application tailored for direct comparison of corresponding histological section images showing PrP and CamKII promoter activity. We report largely complementary distributions of PrP and CamKII promoter activity throughout the entire brain and discuss distributions in selected example regions in more detail. Thus, our online atlas of comprehensive promoter activity pattern in both the Tet-Off PrP and CamKII promoter mouse line will benefit future planning of experiments and goal-directed generation of specific conditional animal models as well as the interpretation of findings from such animals.