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

描述(申请人提供)：5-羟色胺受体(5HT2C)的2C亚型与许多人类精神和行为障碍有关，包括严重抑郁障碍、心境恶劣、强迫症、焦虑和精神分裂症。编码5HT2C受体的转录本可以被多达5个腺苷到肌苷的RNA编辑事件修饰，这个过程负责多达24个受体亚型的细胞特异性表达，这可能代表了一种调节机制，细胞通过改变受体：G蛋白相互作用的有效性和特异性来调节其对细胞外信号的反应。最近的研究发现，在被诊断为焦虑、精神分裂症和抑郁症的患者中，5HT2C的表达发生了变化，这些患者与自杀有关，并对抗抑郁药和抗精神病药物治疗有反应。这项拟议研究的长期目标是确定参与5HT2C表达和信号调节的细胞机制，以及5HT2C编辑和神经精神障碍之间的可能关系。在最近的研究中，我们已经证明了5HT2C mRNA和蛋白质亚型之间的差异，因为只表达完全编辑的5HT2C受体亚型的转基因小鼠表现出前所未有的5HT2C受体密度比野生型动物高40到70倍，而5HT2C mRNA水平保持不变。因此，RNA编辑通过未知的转录后机制影响5HT2C蛋白的表达(S)。这项建议的目的是开发新的转基因工具，通过产生携带盒带受体(CA)等位基因的胚胎干细胞来研究5HT2C受体的许多方面的表达/功能，其中可以通过重组酶介导盒交换引入突变，这一过程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高得多。在这里，我们重点介绍了将表位标签(S)引入内源性5HT2C基因座，从而更有效地纯化5HT2C受体蛋白，以便随后比较不同脑区的mR NA和蛋白质亚型的分布。将通过检测受体表达、配体亲和力和信号的潜在变化来评估在受体内多个位置插入表位在转基因异种细胞系中的功能后果。这个 插入不改变受体功能的表位将被引入小鼠，突变动物将被评估行为、受体表达/功能的变化，以及使用串联亲和层析纯化标记受体的能力。可以预见，这种新型转基因策略的发展不仅将为检测5HT2C基因和蛋白表达的潜在差异提供工具，还将为众多研究人员提供一种更有效的方法，将感兴趣的任何突变(如与疾病相关的SNPs、报告结构、毒素、空/条件等位基因)引入5HT2C受体基因，从而扩大对与5HT2C功能改变相关的人类精神疾病的研究。

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.