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

TKFS使用转基因和条件敲除方法来解决神经生物学中与衰老相关的问题。有两个主要的兴趣领域：I）在发育、药物治疗和氧化应激期间多巴胺（DA）细胞特异性基因表达，和II）理解Mu阿片样物质受体（莫尔）在外周镇痛和免疫应答中的作用。I.通过β-半乳糖苷酶荧光底物标记和从酪氨酸羟化酶- lac Z（THB）转基因小鼠胚胎解剖的磨碎的腹侧中脑组织的FACS分析，分离了胚胎（E14）DA细胞的纯群体。从这些纯化的lac Z+ DA细胞的mRNA被用于构建DA细胞特异性cDNA文库，用于最终鉴定新的DA神经营养因子/受体。在第二项研究中，为了寻找DA细胞特异性基因表达元件，将多巴胺转运蛋白（DAT）近端启动子序列（高达-2.8Kb）融合到lac Z并引入转基因小鼠中。原位B-gal染色已经在蓝斑中产生了来自4个DAT-lac Z构建体中的每一个的多个创始细胞系的意外表达，没有鉴定到DA细胞表达。二. Cre-loxp策略被用于条件性敲除外周神经元和T淋巴细胞中的莫尔，以建立新的炎症模型来研究疼痛和免疫应答。该模型包括侧接莫尔基因外显子3的loxp位点插入（MULX），通过外周蛋白-Cre（PCRE）转基因在背根神经节（DRG）中的Cre表达，以及先前表征的T细胞特异性LCK-CRE小鼠（来自Jamey马斯）。MULX和PCRE小鼠目前分别表征了莫尔和CRE表达。所得到的敲除小鼠应有助于确定Mu、Delta和Kappa激动剂的外周镇痛作用中莫尔的作用，并有助于最终治疗疼痛，而没有吗啡的中枢阿片样物质副作用，如成瘾和欣快。- Mu阿片受体;多巴胺转运体; cre; lox; FACS; lac Z;镇痛;炎症;免疫;背根神经节;吗啡

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