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.