Novel transgenic tools for analysis of 5HT2C receptor expression and function

用于分析 5HT2C 受体表达和功能的新型转基因工具

• 批准号: 8299772
• 负责人: Ronald B. Emeson
• 金额: $ 19.48万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2014-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8299772
• 关键词: Adenosine; Affinity; Affinity Chromatography; Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly; Alleles; Animals; Antidepressive Agents; Antipsychotic Agents; Ants; Anus; Anxiety; Behavior; Behavior Disorders; Behavioral; Brain region; Cell Line; Cells; Cognitive deficits; Complex; Coupling; DNA; Development; Diagnosis; Disease; Dysthymic Disorder; Epitopes; Event; Exhibits; Failure to Thrive; Fibroblasts; GTP-Binding Proteins; Generations; Genes; Growth; Human; Hyperphagia; Hypogonadism; Inosine; Ligands; Major Depressive Disorder; Mediating; Mental Depression; Mental disorders; Messenger RNA; Metals; Methods; Morbid Obesity; Mus; Muscle hypotonia; Mutant Strains Mice; Mutation; Nervous System Physiology; Neuraxis; Neuroblastoma; Patients; Pattern; Phenocopy; Phenotype; Physiological; Positioning Attribute; Prader-Willi Syndrome; Process; Property; Protein Isoforms; Proteins; RNA Editing; Receptor Gene; Regulation; Reporter; Research; Research Personnel; Schizophrenia; Serotonin; Signal Transduction; Site; Specificity; Suicide; System; Toxin; Transcript; Transgenic Organisms; United States National Institutes of Health; addiction; brain tissue; embryonic stem cell; extracellular; homologous recombination; imprint; infancy; interest; mRNA Expression; mutant; neuropsychiatry; novel; protein expression; receptor; receptor density; receptor expression; receptor function; recombinase; response; serotonin receptor; tool; tool development

DESCRIPTION (provided by applicant): The 2C-subtype of serotonin receptor (5HT2C) has been implicated in a number of human psychiatric and behavioral disorders, including major depressive disorder, dysthymia, obsessive-compulsive disease, anxiety, and schizophrenia. Transcripts encoding the 5HT2C receptor can be modified by up to five adenosine-to-inosine RNA editing events, a process responsible for the cell-specific expression of as many as 24 receptor isoforms which may represent a regulatory mechanism by which cells modulate their response to extracellular signals by altering the efficacy and specificity of receptor:G-protein interactions. More recent studies have identified alterations in 5HT2C expression in patients diagnosed with anxiety, schizophrenia and depression associated with suicide and in response to antidepressant and antipsychotic treatment. The long term objectives of the proposed research are to define the cellular mechanisms involved in the regulation of 5HT2C expression and signaling, as well as possible relationships between 5HT2C editing and neuropsychiatric disorders. In re- cent studies, we have demonstrated a disparity between 5HT2C mRNA and protein isoforms as genetically modified mice solely expressing the fully edited isoform of the 5HT2C receptor exhibit an unprecedented 40- to 70-fold increase in 5HT2C receptor density compared to wild-type animals, yet 5HT2C mRNA levels remain un- changed. Thus, RNA editing has dramatic consequences on the expression of 5HT2C protein through uncharacterized post-transcriptional mechanism(s). The objectives of this proposal are to develop novel transgenic tools to investigate numerous aspects of 5HT2C receptor expression/function by the generation of embryonic stem cells harboring a Cassette Acceptor (CA) allele in which mutations may be introduced using recombinase mediated cassette exchange, a process that can rapidly and efficiently insert different DNA fragments into specific gene loci and is significantly more efficient than homologous recombination. Here we focus upon the introduction of epitope tag(s) into the endogenous 5HT2C locus, allowing a more effective purification of 5HT2C receptor protein for subsequent comparisons of mRNA and protein isoform distribution in discrete brain regions. The functional consequences of epitope insertion at multiple sites within the receptor will be assessed in transfected heterologous cell lines by examining potential alterations in receptor expression, ligand affinity and signaling. The insertion of an epitope that does not alter receptor function will be introduced into mice and mutant animals will be assessed for alterations in behavior, receptor expression/function and the ability to purify the tagged receptor using tandem affinity chromatography. It is anticipated that the development of this novel transgenic strategy will not only provide tools to examine potential disparities in 5HT2C mRNA and protein expression, but also provide numerous researchers with a more efficient method to introduce any mutation of interest (e.g. disease-associated SNPs, reporter constructs, toxins, null/conditional alleles) into the 5HT2C receptor gene, thus expanding research into human psychiatric disorders related to altered 5HT2C function. PUBLIC HEALTH RELEVANCE: The proposed studies will allow the development of novel transgenic tools to increase our ability to examine the functional diversity and mechanisms regulating the expression and function of serotonin 2C (5HT2C) receptors in the mammalian central nervous system. The introduction of epitope tag(s) into the receptor and the development of embryonic stem cells harboring Cassette Acceptor (CA) alleles provide a facile strategy by which investigators can generate mice containing mutations within this receptor using recombinase mediated cassette exchange and circumvent the low-targeting efficiency generally associated with homologous recombination in embryonic stem cells. The development of these tools has the potential to assist numerous investigators in their research, diagnosis and treatment of neuropsychiatric disorders such as schizophrenia, depression, anxiety and addiction, where alterations in 5HT2C receptor expression and function have been implicated.

描述（由申请人提供）：5-羟色胺受体（5 HT 2C）的2C亚型与许多人类精神和行为障碍有关，包括重度抑郁症、心境恶劣、强迫症、焦虑症和精神分裂症。编码5 HT 2C受体的转录物可以通过多达五个腺苷至肌苷RNA编辑事件进行修饰，这是一个负责多达24种受体亚型的细胞特异性表达的过程，这可能代表了一种调节机制，细胞通过改变受体：G蛋白相互作用的功效和特异性来调节其对细胞外信号的反应。最近的研究已经确定了诊断为焦虑症、精神分裂症和抑郁症的患者中5 HT 2C表达的改变，这些患者与自杀有关，并且对抗抑郁药和抗精神病药治疗有反应。这项研究的长期目标是确定参与5 HT 2C表达和信号传导调控的细胞机制，以及5 HT 2C编辑和神经精神疾病之间的可能关系。在最近的研究中，我们已经证明了5 HT 2C mRNA和蛋白质同种型之间的差异，因为与野生型动物相比，仅表达完全编辑的5 HT 2C受体同种型的遗传修饰小鼠表现出前所未有的5 HT 2C受体密度增加40至70倍，但5 HT 2C mRNA水平保持不变。因此，RNA编辑通过未表征的转录后机制对5 HT 2C蛋白的表达具有显著影响。该提议的目的是开发新的转基因工具，以通过产生携带盒受体（CA）等位基因的胚胎干细胞来研究5 HT 2C受体表达/功能的许多方面，其中可以使用重组酶介导的盒交换引入突变，一种可以快速有效地将不同DNA片段插入特定基因位点的过程，重组在这里，我们专注于引入表位标签（S）到内源性5 HT 2C基因座，允许更有效的纯化5 HT 2C受体蛋白的mRNA和蛋白质亚型分布在离散的脑区域的后续比较。通过检查受体表达、配体亲和力和信号传导的潜在改变，在转染的异源细胞系中评估表位插入受体内多个位点的功能后果。的 将不改变受体功能的表位的插入引入小鼠，并评估突变动物的行为、受体表达/功能的改变和使用串联亲和色谱纯化标记受体的能力。预期这种新的转基因策略的发展将不仅提供检查5 HT 2C mRNA和蛋白质表达的潜在差异的工具，而且还为许多研究人员提供引入任何感兴趣的突变的更有效的方法（例如疾病相关SNP、报告构建体、毒素、无效/条件等位基因）插入5 HT 2C受体基因，从而扩展了对与5 HT 2C功能改变相关的人类精神疾病的研究。 公共卫生相关性：拟议的研究将允许开发新的转基因工具，以提高我们的能力，检查功能多样性和机制，调节5-羟色胺2C（5 HT 2C）受体在哺乳动物中枢神经系统的表达和功能。将表位标签引入受体和开发携带盒受体（CA）等位基因的胚胎干细胞提供了一种简单的策略，通过该策略，研究者可以使用重组酶介导的盒交换产生在该受体内含有突变的小鼠，并避免通常与胚胎干细胞中的同源重组相关的低靶向效率。这些工具的开发有可能帮助许多研究人员研究，诊断和治疗神经精神疾病，如精神分裂症，抑郁症，焦虑症和成瘾，其中5 HT 2C受体表达和功能的改变已被牵连。