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

TKFS使用转基因和条件基因敲除方法来解决神经生物学中与衰老有关的问题。感兴趣的两个领域是：i)多巴胺(DA)神经元在发育、药物治疗和神经毒性过程中的基因表达；ii)了解Mu阿片受体(MOR)在外周镇痛和免疫反应中的作用。一、纯化胚胎(E13.5)DA神经元，分离mRNA，构建E13.5 DA神经元基因文库。酪氨酸羟基酶-LacZ(THB)转基因小鼠胚胎在胚胎腹侧中脑DA神经元表达β-半乳糖苷酶。这些细胞被标记有β-半乳糖苷酶的荧光底物，并通过荧光激活的细胞分选进行纯化。对960个DA基因进行部分DNA序列分析，构建DA微阵列。对DA神经元微阵列和NIA神经阵列(由1100个在神经系统表达的人类cDNA组成)上的腹侧中脑RNA和DA cDNAs的微阵列分析，提供了超过100个新的EST和大量胚胎增强型中脑腹侧cDNA的初步DA神经元基因表达谱。甲基苯丙胺神经毒性的微阵列分析显示，治疗12小时后，腹侧中脑中Coxi的表达增加了2倍以上。在一项寻找DA细胞特异性基因表达元件的平行研究中，将多巴胺转运蛋白基因(DAT)近端启动子序列(高达-2.8Kb)与lac Z融合并导入转基因小鼠。原位β-半乳糖苷酶染色在蓝斑中发现了来自4个DAT-LacZ结构的多个方正细胞系的意外表达，没有发现DA细胞特异性的表达。使用Cre-loxP策略有条件地敲除外周神经元和T淋巴细胞中的MOR，以建立一种新的小鼠炎症模型来研究疼痛和免疫反应。该模型包括在MOR基因外显子3两侧插入loxP位点，通过外周蛋白-Cre(PCRE)转基因在背根神经节(DRG)表达Cre，以及在先前描述的LCK-Cre小鼠(Jamey Marth)中表达T细胞特异性Cre。用Rosa Cre报告株对PCRE小鼠的5个建立系进行了分析，发现Cre在周围神经系统中有表达，在DRG中有高表达。由此产生的基因敲除小鼠应该有助于阐明MOR与其他阿片受体(Kappa和Delta)在外周止痛和炎症中的作用。插入loxP的MOR基因小鼠仍在构建中，但对本工作中发现的C57BL/6和129/Sv MOR基因组序列的品系间差异进行比较发现，许多多态可能有助于品系对疼痛刺激和阿片类药物的特异性反应。这些发现暗示了这些反应中人类个体间变异的潜在遗传来源。

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.