Inducible recombination and gene expression in specific neurotransmitter systems

特定神经递质系统中的诱导重组和基因表达

• 批准号: 8225329
• 负责人: STEVEN A THOMAS
• 金额: $ 35.08万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8225329
• 关键词: 3' Untranslated Regions; Adrenergic Agents; Adult; Aftercare; Animal Model; Antibiotics; Anxiety; Arousal; Attention; Bacterial Artificial Chromosomes; Behavioral; Catecholamines; Chimeric Proteins; Cognitive; Communities; Complementary DNA; Development; Disease; Dopamine; Doxycycline; Drug abuse; Encephalomyocarditis virus; Enterobacteria phage P1 Cre recombinase; Enzymes; Epinephrine; Estrogen Receptors; Etiology; Gene Expression; Gene Targeting; Gene-Modified; Genes; Genetic Recombination; Genetic Transcription; Goals; Health; High Pressure Liquid Chromatography; Immunofluorescence Immunologic; Internal Ribosome Entry Site; Learning; Ligand Binding Domain; Link; Location; Mediating; Memory; Mental Depression; Mental disorders; Messenger RNA; Mixed Function Oxygenases; Modification; Monitor; Motivation; Mus; Narcolepsy; Neurons; Neurosciences; Neurotransmitters; Norepinephrine; Pattern; Pharmaceutical Preparations; Physiological; Post-Traumatic Stress Disorders; Process; Proteins; Receptor Signaling; Reporter; Research; Research Personnel; Rewards; Role; Serotonin; Site; Sleep; Specific qualifier value; Stress; System; Tamoxifen; Tetanus Helper Peptide; Tetracyclines; Time; Trans-Activators; Transgenes; Transgenic Organisms; Tryptophan 5-monooxygenase; Tyrosine 3-Monooxygenase; adrenergic; base; dopamine system; dopamine transporter; dopaminergic neuron; embryonic stem cell; gene cloning; gene function; homologous recombination; hypocretin; insight; interest; mouse model; neurophysiology; norepinephrine system; programs; promoter; research study

DESCRIPTION (provided by investigator): The goal of this proposal is to advance our ability to inducibly manipulate gene expression within subsets of neurons. Four neurotransmitter systems have been chosen for this proposal based on their having been implicated in numerous neurophysiologic and behavioral processes, as well as disorders of those processes. The four are the adrenergic (norepinephrine and epinephrine), dopaminergic, serotonergic and orexinergic systems. Because of the unique ability to create gene-targeted mice among mammals, mice will be the model organism employed. One goal is to inducibly activate, inactivate or modify endogenous genes that have been endowed with unique loxP sites that are recognized by bacteriophage P1 Cre recombinase (Cre). The main approach for achieving this will be the neurotransmitter-specific expression of the tamoxifen-inducible Cre fusion protein, CreERT2, that is fused to a modified estrogen receptor ligand-binding domain (ERT2). Because there may be occasions when using tamoxifen should be avoided due to effects on endogenous estrogen receptor signaling, a second approach for achieving inducible Cre activity will also be pursued. For this approach, the tetracycline-inducible transactivator (tTA2) and reverse tTA (rtTA2S-M2) will be expressed specifically in each neurotransmitter system. When crossed with previously characterized tetO-Cre mice, the transactivator (TA) mice will induce the neurotransmitter-specific expression of Cre either in the absence (tTA) or in the presence (rtTA) of antibiotic. In addition, the TA mice will permit the induction of any other Ptet-cDNA transgene in a neurotransmitter-specific fashion. To obtain neurotransmitter-specific expression, genes that uniquely define these neurotransmitter systems will be utilized: for the adrenergic system - dopamine 2- hydroxylase, for the dopaminergic system - dopamine transporter, for the serotonergic system - tryptophan hydroxylase 2, and for the orexinergic system - orexin. CreERT2 and the TAs will be targeted to the 3'- untranslated region of each gene via homologous recombination in embryonic stem cells. To achieve bicistronic expression, CreERT2 and the TAs will be preceded by an internal ribosome entry site. Reporter mice for Cre activity will be used to correlate temporal and spatial expression patterns of Cre with those for the targeted endogenous gene. Finally, the utility of the CreERT2 and TA mice will be demonstrated by crossing them with mice harboring a floxed tyrosine hydroxylase gene for inducible transmitter depletion. PUBLIC HEALTH RELEVANCE The study of these four neurotransmitter systems via inducible changes in gene expression that are specific to each system is highly relevant to the understanding of fundamental neurophysiological and behavioral processes such as sleep, arousal and attention, motivation and reward, and learning and memory, as well as disorders related to these processes that include narcolepsy, drug abuse, anxiety, depression and post- traumatic stress disorder. It is expected that the proposed mouse models will permit significant insights into the etiology, identification and treatment of these disorders.

描述（研究人员提供）：该提案的目的是提高我们在神经元子集中操纵基因表达的能力。 该提案已选择了四个神经递质系统，这些系统与众多神经生理学和行为过程有关，以及这些过程的疾病。 这四种是肾上腺素能（去甲肾上腺素和肾上腺素），多巴胺能，血清素能和甲状腺素能系统。 由于具有在哺乳动物中创建基因靶向小鼠的独特能力，因此将是使用的模型生物。 一个目标是诱导诱导，灭活或修饰的内源基因，这些基因已赋予了独特的LOXP位点，这些基因被噬菌体P1 CRE重组酶（CRE）识别。 实现此目的的主要方法是他莫昔芬诱导的Cre融合蛋白Creert2的神经递质特异性表达，它与改良的雌激素受体配体结合结构域（ERT2）融合在一起。 由于可能有时应避免使用他莫昔芬，这是由于对内源性雌激素受体信号传导的影响，因此还将采用第二种实现诱导型CRE活性的方法。 对于这种方法，四环素诱导的反式激活器（TTA2）和反向TTA（RTTA2S-M2）将在每个神经递质系统中专门表达。 当与先前表征的TETO-CRE小鼠交叉时，反式激活器（TA）小鼠将在不存在（TTA）或抗生素（RTTA）的情况下诱导CRE的神经递质特异性表达。 此外，TA小鼠将允许以神经递质特异性的方式诱导任何其他PTET-CDNA转基因。 为了获得神经递质特异性表达，将使用唯一定义这些神经递质系统的基因：对于肾上腺素能系统 - 多巴胺能系统 - 多巴胺能系统 - 多巴胺系统 - 多巴胺转运蛋白，用于血清素剂系统 - 尖锐甲基氧基羟基氢氧化氢酶2和systance orex orex orex -orex orex -orex -orex orex。 CREERT2和TAS将通过胚胎干细胞中的同源重组靶向每个基因的3'-未翻译区域。 为了实现双科体的表达，CREERT2和TAS将在内部核糖体进入位点之前。 用于CRE活性的记者小鼠将使用CRE的时间和空间表达模式与靶向内源基因相关。 最后，将Creert2和TA小鼠的效用通过与携带抗诱导发射机耗竭的抗酪氨酸羟化酶基因的小鼠交叉来证明它们。 PUBLIC HEALTH RELEVANCE The study of these four neurotransmitter systems via inducible changes in gene expression that are specific to each system is highly relevant to the understanding of fundamental neurophysiological and behavioral processes such as sleep, arousal and attention, motivation and reward, and learning and memory, as well as disorders related to these processes that include narcolepsy, drug abuse, anxiety, depression and post- traumatic stress disorder. 预计所提出的小鼠模型将允许对这些疾病的病因，鉴定和治疗有重大见解。