TISSUE SPECIFIC EXPRESSION OF DAT & MOR TRANSGENES IN THE STUDY OF AGING DISORDER

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The TKFS uses transgenic and conditional knockout approaches to address aging related questions in neurobiology. There are two main areas of interest: I) dopamine (DA) cell specific gene expression during development, drug treatment, and oxidative stress, and II) an understanding of the role of the Mu Opioid Receptor (MOR)in peripheral analgesia and immune response. I. A pure population of embryonic (E14) DA cells has been isolated via B-galactosidase fluorescent substrate labeling and FACS analysis of triturated ventral midbrain tissue dissected from tyrosine hydroxylase - lac Z (THB)transgenic mouse embryos. mRNA from these purified lac Z+ DA cells is being utilized to construct a DA cell specific cDNA library for the eventual identification of novel DA neurotrophic factors/receptors. In a second study, in search of DA cell specific gene expression elements, the dopamine transporter (DAT) proximal promoter sequences(up to -2.8Kb) were fused to lac Z and introduced into transgenic mice. In situ B-gal staining has yielded unexpected expression in the locus coeruleus for multiple founder lines from each of 4 x DAT - lac Z constructs, with no DA cell expression identified. II. A Cre-loxp strategy is being employed to conditionally knockout the MOR in peripheral neurons and T lymphocytes in the creation of a new inflammation model to study pain and the immune response. This model includes loxp site insertions flanking exon 3 of the MOR gene (MULX), Cre expression in the Dorsal Root Ganglion (DRG) via a Peripherin-Cre (PCRE) transgene, and the previously characterized T-cell specific LCK-CRE mouse(from Jamey Marth). Both MULX and PCRE mice are currently being characterized for MOR and CRE expression, respectively. The resulting knockout mouse should help define the role of the MOR in the peripheral analgesic effects of Mu, Delta and Kappa agonists, and lend itself to the eventual treatment of pain without the central opioid side effects of morphine, such as addiction and euphoria. - Mu Opioid Receptor; Dopamine transporter; cre; lox; FACS; lac Z; analgesia; inflammation; immune; dorsal root ganglion; morphine