Analysis of Enhanced Green Fluorescent Protein Expression in Corticotropin-Releasing Hormone Neurons in CRH-eGFP BAC Transgenic Mice
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Corticotropin-releasing hormone (CRR) is the major hypothalamic regulator of the mammalian stress response. It is expressed at numerous sites in the brain to control the endocrine, autonomic, and behavioral responses to stress, and dysregulation of CRH activity contributes to major depression and anxiety disorders. Studies to characterize the regulatory properties of CRH neurons in the brain would be greatly enhanced by the targeting of an observable marker such as enhanced green fluorescent protein (eGFP) specifically in CRH neurons. Therefore Seasholtz et al. introduced eGFP by homologous recombination into two different bacterial artificial chromosomes (BACs) containing the mouse CRH gene, and the modified CRH-eGFP BACs were used to create transgenic mice. In these mice, the expression of eGFP should be directed by the entire CRH gene locus including large regions of 5' and 3' CRH flanking DNA. It was hypothesized that these sequences would direct eGFP expression only in CRH--expressing cells. The CRH-eGFP BAC transgenic mice were evaluated using reverse transcriptase-PCR within different brain regions. The CRH-eGFP SAC transgenic lines were also evaluated using immunocytochemistry and in situ hybridization histochemistry. Low level eGFP immunofluorescence was detected in numerous brain regions where CRH is highly expressed; to support this CRH mRNA was also detected in sequential sections of tissue. Data suggest that eGFP expression is usually limited to CRH-expressing neurons, but that not all CRH neurons express detectable levels of eGFP. Additional enhancer elements may be necessary for increased expression of eGFP in CRH neurons in CRH-eGFP SAC mice.