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）：血清素受体（5HT2C）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 5HT2C 受体的转录本可通过多达 5 个腺苷至肌苷 RNA 编辑事件进行修改，该过程负责多达 24 种受体亚型的细胞特异性表达，这可能代表细胞调节其响应的调节机制。通过改变受体：G 蛋白相互作用的功效和特异性来产生细胞外信号。最近的研究发现，被诊断患有与自杀相关的焦虑、精神分裂症和抑郁症以及抗抑郁药和抗精神病药治疗反应的患者中 5HT2C 表达发生变化。拟议研究的长期目标是确定参与 5HT2C 表达和信号传导调节的细胞机制，以及 5HT2C 编辑与神经精神疾病之间的可能关系。在最近的研究中，我们证明了 5HT2C mRNA 和蛋白质同工型之间的差异，因为仅表达完全编辑的 5HT2C 受体同工型的转基因小鼠与野生型相比，5HT2C 受体密度史无前例地增加了 40 至 70 倍。动物中，但 5HT2C mRNA 水平保持不变。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影响。该提案的目标是开发新型转基因工具，通过生成携带盒受体（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，其中可以使用重组酶介导的盒交换（这一过程）引入突变它可以快速有效地将不同的DNA片段插入特定的基因位点，并且比同源重组效率明显更高。在这里，我们重点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，以便随后比较离散脑区域中 mRNA 和蛋白质异构体的分布。通过检查受体表达、配体亲和力和信号传导的潜在变化，将在转染的异源细胞系中评估受体内多个位点插入表位的功能后果。这不改变受体功能的表位插入将被引入小鼠中，并且将评估突变动物的行为、受体表达/功能的改变以及使用串联亲和层析纯化标记受体的能力。预计这种新型转基因策略的开发不仅将提供检查 5HT2C mRNA 和蛋白质表达中潜在差异的工具，而且还为众多研究人员提供更有效的方法来引入任何感兴趣的突变（例如与疾病相关的 SNP、报告构建体、毒素、无效/条件等位基因）到5HT2C受体基因中，从而扩大了对与5HT2C功能改变相关的人类精神疾病的研究。公共健康相关性：拟议的研究将允许开发新型转基因工具，以提高我们检查哺乳动物中枢神经系统中血清素 2C (5HT2C) 受体表达和功能调节功能多样性和机制的能力。将表位标签引入受体以及携带盒式受体（CA）等位基因的胚胎干细胞的发育提供了一种简便的策略，通过该策略，研究人员可以使用重组酶介导的盒式交换来产生含有该受体内突变的小鼠，并规避低-靶向效率通常与胚胎干细胞中的同源重组相关。这些工具的开发有可能帮助众多研究人员研究、诊断和治疗精神分裂症、抑郁症、焦虑症和成瘾等神经精神疾病，这些疾病与 5HT2C 受体表达和功能的改变有关。