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

基本信息

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
项目类别：
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

项目摘要

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（5HT2C）的2C-亚型与许多人类的精神病和行为障碍有关，包括重大抑郁症，心律失常，强迫症，强迫性疾病，焦虑，焦虑和精神分裂症。编码5HT2C受体的转录本可以通过多达五个腺苷对肌苷RNA编辑事件进行修饰，这是一个导致多达24种受体同工型细胞特异性表达的过程，该过程可能代表一种调控机制，该机制可以通过该机制调节其对细胞外信号的反应，从而改变受体的效率和特异性受体相互作用：Gorpoteins：Gorpoteions：Gorpoteions。最新的研究已经确定了5HT2C表达的改变，该患者被诊断出患有焦虑症，精神分裂症和抑郁症以及对抗抑郁药和抗精神病药的反应。拟议研究的长期目标是定义与5HT2C表达和信号传导调控有关的细胞机制，以及5HT2C编辑与神经精神疾病之间的可能关系。在重点研究中，我们证明了5HT2C mRNA和蛋白质同工型之间的差异是因为仅表达5HT2C受体的完全编辑的同工型的遗传改性小鼠表现出前所未有的40至70倍，与野生型动物相比，5HT2C受体密度在5HT2C受体密度中增加了，但仍然更改了5HHTTYPE级别。因此，RNA编辑通过未表征的转录后机制对5HT2C蛋白的表达产生巨大后果。该提案的目标是开发新型的转基因工具，通过产生具有盒式录音带受体（CA）等位基因的胚胎干细胞（CA）等位基因的胚胎干细胞来研究5HT2C受体表达/功能的许多方面，在该胚胎中，可以使用重组酶介导的盒式盒交换来引入突变，这一过程可以快速地插入不同的DNA片段，而不是更有效地将不同的DNA Recompin插入特定的基因组合。在这里，我们关注将表位标签引入内源性5HT2C基因座，从而更有效地纯化5HT2C受体蛋白，以便在离散大脑区域中对mRNA和蛋白质同工型的随后比较。表位插入在受体内部多个位点的功能后果将通过检查受体表达，配体亲和力和信号传导的潜在变化来评估在转染的异源细胞系中。这将插入不改变受体功能的表位将被引入小鼠，并将评估突变动物的行为，受体表达/功能以及使用串联亲和色谱净化标记的受体的能力。 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功能。公共卫生相关性：拟议的研究将允许开发新型的转基因工具，以提高我们检查哺乳动物中枢神经系统中羟色胺2C（5HT2C）受体的表达和功能的功能多样性和机制的能力。将表位标签引入受体和具有盒式磁带受体（CA）等位基因的胚胎干细胞的发育提供了一种轻松的策略，通过该策略，研究人员可以通过该策略来通过该策略生成该受体中含有突变的小鼠，并使用重物组织介导的盒式盒式磁带交换并绕过与胚胎干细胞中的同源物质重组相关的低含量效应。这些工具的开发有可能协助众多研究人员的研究，诊断和治疗神经精神疾病，例如精神分裂症，抑郁症，焦虑和成瘾，其中涉及5HT2C受体表达和功能的改变。