Inducible recombination and gene expression in specific neurotransmitter systems

描述（由研究者提供）：本提案的目标是提高我们在神经元亚群中诱导操纵基因表达的能力。本研究选择了四种神经递质系统，因为它们与许多神经生理和行为过程以及这些过程的紊乱有关。这四个系统是肾上腺素能系统（去甲肾上腺素和肾上腺素）、多巴胺能系统、血清素能系统和食欲能系统。由于在哺乳动物中创造基因靶向小鼠的独特能力，小鼠将成为被采用的模式生物。一个目标是诱导激活、灭活或修饰内源性基因，这些基因被赋予了独特的loxP位点，可以被噬菌体P1 Cre重组酶（Cre）识别。实现这一目标的主要方法将是他莫昔芬诱导的Cre融合蛋白CreERT2的神经递质特异性表达，该融合蛋白与修饰的雌激素受体配体结合域（ERT2）融合。由于它莫西芬对内源性雌激素受体信号的影响，可能在某些情况下应避免使用它莫西芬，因此还将寻求实现诱导Cre活性的第二种方法。对于这种方法，四环素诱导的转激活子（tTA2）和反向tTA （rtTA2S-M2）将在每个神经递质系统中特异性表达。当与先前表征的tetO-Cre小鼠杂交时，transactivator （TA）小鼠在不存在（tTA）或存在（rtTA）抗生素的情况下都会诱导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pet - cdna转基因。为了获得神经递质特异性表达，将利用定义这些神经递质系统的独特基因：肾上腺素能系统-多巴胺2-羟化酶，多巴胺能系统-多巴胺转运蛋白，血清素能系统-色氨酸羟化酶2，以及食欲素能系统-食欲素。在胚胎干细胞中，通过同源重组，CreERT2和TAs将靶向每个基因的3'-非翻译区。为了实现双电子表达，CreERT2和TAs将在内部核糖体进入位点之前进行。Cre活性报告小鼠将用于将Cre的时空表达模式与目标内源基因的表达模式联系起来。最后，将通过将CreERT2和TA小鼠与携带固定酪氨酸羟化酶基因的小鼠杂交，以诱导递质耗用，从而证明它们的效用。对这四种神经递质系统的研究，通过对每个系统特异性的基因表达的诱导变化，与理解基本的神经生理和行为过程（如睡眠、觉醒和注意力、动机和奖励、学习和记忆）以及与这些过程相关的疾病（包括嗜睡症、药物滥用、焦虑、抑郁和创伤后应激障碍）高度相关。预计所提出的小鼠模型将对这些疾病的病因、识别和治疗提供重要的见解。

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.