Analysis of enhanced Green Fluorescent Protein Expression in Corticotropin-Releasing Hormone Neurons in CRH-eGFP BAC Transgenic Mice

Abstract

The endocrine response to stress is mediated by the hypothalamic-pituitary-adrenal (HPA) axis (Figure 1). The key hypothalamic regulator within the HPA axis is corticotropin-releasing hormone (CRH). Dysregulation within the stress axis has been linked to the development of depression and anxiety disorders in humans, and high blood concentrations of CRH have been observed in depressed patients. Studies to characterize the regulatory properties of CRH neurons in the brain would be greatly enhanced by the targeting of an observable marker specifically in CRH neurons. In previous studies, Keegan and colleagues created transgenic mouse models using different regions of the CRH promoter and genomic sequences to direct expression of the beta-galactosidase reporter gene. In these studies, numerous lines exhibited little to no detectable beta-galactosidase expression in sites of CRH expression and exhibited sites of ectopic CNS expression. It was postulated that the absence of essential cisacting enhancer sequences at significant distances (greater than 50 kb) were responsible for the lack of cell-specific and temporally controlled reporter expression localized to CRH expressing cells. The recent use of bacterial artificial chromosomes (BACs) containing 100 -300 kb of genomic DNA has allowed numerous groups to direct accurate in vivo cell specific expression of reporter genes. Seasholtz et al. used this technology to create various lines of transgenic mice from two CRH BACs (BAC RP23-441G11 and BAC RP23- 129A14)(Figure 2). These lines hypothetically express enhanced green fluorescent protein (eGFP) under the control of the entire CRH gene locus including large regions of 5’ and 3’ CRH flanking DNA. The data shown here document the eGFP expression in multiple transgenic founder lines obtained from homologous recombination in two different CRH BAC constructs in order to determine if these constructs are being expressed specifically in CRH expressing neurons.