Dopamine neuron molecular phenotype and Cre-loxP knockout of mu opioid receptor

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。 感兴趣的两个领域是：I）在发育、药物治疗和神经毒性期间多巴胺（DA）神经元基因表达，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.纯化胚胎（E13.5）DA神经元，分离mRNA，并构建E13.5 DA神经元cDNA文库。酪氨酸羟化酶- lacZ（THB）转基因小鼠胚胎在胚胎腹侧中脑DA神经元中表达β-半乳糖苷酶。 用β-半乳糖苷酶的荧光底物标记这些细胞，并通过荧光激活细胞分选进行纯化。 对960个DA cDNA进行部分DNA序列分析，并用于创建DA微阵列。 在DA神经元微阵列和NIA神经阵列（由在神经系统中表达的1100个人cDNA组成）上的腹侧中脑RNA和DA cDNA的微阵列分析提供了具有超过100个新的EST和许多胚胎增强的腹侧中脑cDNA的初始DA神经元基因表达谱。 甲基苯丙胺神经毒性的微阵列分析揭示了在处理后12小时，腹侧中脑中COXI表达增强超过2倍。 在寻找DA细胞特异性基因表达元件的平行研究中，多巴胺转运蛋白基因（DAT）近端启动子序列（高达-2.8Kb）与lac Z融合并引入转基因小鼠。 原位β-半乳糖苷酶染色已经在蓝斑中产生了来自4个DAT-lacZ构建体中的每一个的多个创始细胞系的意想不到的表达，没有鉴定出DA细胞特异性表达。 二. 采用条件性敲除外周神经元和T淋巴细胞中的莫尔的Cre-loxP策略来创建新的炎症小鼠模型以研究疼痛和免疫应答。 该模型包括莫尔基因外显子3侧翼的loxP位点插入、通过外周蛋白-Cre（PCRE）转基因在背根神经节（DRG）中的Cre表达、以及在先前表征的LCK-CRE小鼠（Jamey马斯）中的T细胞特异性Cre表达。 已经用ROSA Cre报告菌株分析了PCRE小鼠的五个创始系，其揭示了Cre在外周神经系统中的表达，在DRG中具有高表达。 由此产生的基因敲除小鼠应该有助于阐明莫尔与其他阿片受体（κ和δ）在外周镇痛和炎症中的作用。 loxP插入的莫尔基因小鼠仍在构建中，但在这项工作中鉴定的C57 BL/6和129/Sv莫尔基因组序列之间的品系间差异的比较揭示了许多多态性，这些多态性可能有助于品系特异性对疼痛刺激和阿片类药物的反应。 这些研究结果表明，这些反应的个体间人类变异性的潜在遗传来源。

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.