The TKFS uses transgenic and conditional knockout approaches to address aging related questions in neurobiology. The two areas of interest are: I) dopamine (DA) neuron gene expression during development, drug treatment, and neurotoxicity, and II) an understanding of the role of the Mu Opioid Receptor (MOR) in peripheral analgesia and immune response. I. Embryonic (E13.5) DA neurons have been purified, mRNA isolated, and an E13.5 DA neuron cDNA library constructed. Tyrosine hydroxylase - lacZ (THB) transgenic mouse embryos express beta-galactosidase in embryonic ventral midbrain DA neurons. These cells were tagged with fluorescent substrates for beta-galactosidase and purified via fluorescent activated cell sorting. 960 DA cDNAs were subjected to partial DNA sequence analysis and used to create a DA microarray. Microarray analysis of ventral midbrain RNA and DA cDNAs on both the DA neuron microarray and the NIA neuro-array (composed of 1100 human cDNAs expressed in the nervous system), provides an initial DA neuron gene expression profile with over 100 new ESTs and numerous embryonic enhanced ventral midbrain cDNAs. Microarray analysis of methamphetamine neurotoxicity reveals greater than 2 fold enhancement of COXI expression in ventral midbrain at 12 hours post treatment. In a parallel study to search for DA cell-specific gene expression elements, the dopamine transporter gene (DAT) proximal promoter sequences (up to -2.8Kb) were fused to lac Z and introduced into transgenic mice. In situ beta-galactosidase staining has yielded unexpected expression in the locus coeruleus for multiple founder lines from each of 4 x DAT - lacZ constructs, with no DA cell-specific expression identified. II. A Cre-loxP strategy to conditionally knockout the MOR in peripheral neurons and T lymphocytes is employed to create a new mouse model of inflammation to study pain and the immune response. This model includes loxP site insertions flanking exon 3 of the MOR gene, Cre expression in the Dorsal Root Ganglion (DRG) via a Peripherin-Cre (PCRE) transgene, and T-cell specific Cre expression in the previously characterized LCK-CRE mouse (Jamey Marth). Five founder lines of PCRE mice have been analyzed with the ROSA Cre reporter strain that reveal Cre expression in the peripheral nervous system with high expression in the DRG. The resulting knockout mouse should help clarify the role of the MOR vs the other opioid receptors (kappa and delta) in peripheral analgesia and inflammation. The loxP inserted MOR gene mouse is still under construction, but a comparison of the inter-strain differences between the C57BL/6 and 129/Sv MOR genomic sequences identified in this work reveals many polymorphisms that might contribute to strain-specific responses to painful stimuli and opiate drugs. These findings suggest potential genetic sources of inter-individual human variability in these responses.
