Analysis of Enhanced Green Fluorescent Protein Expression in Corticotropin-Releasing Hormone Neurons in CRH-eGFP BAC Transgenic Mice
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Authors
Cregan, Lindsey
Issue Date
2006
Type
Thesis
Language
en_US
Keywords
Alternative Title
Abstract
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.
Description
v, 32 p.
Citation
Publisher
Kalamazoo College
License
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