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

描述（由申请人提供）：5 -羟色胺受体2c亚型（5HT2C）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受体的转录本可以通过多达五种腺苷-肌苷RNA编辑事件进行修饰，这一过程负责多达24种受体同种异构体的细胞特异性表达，这可能代表了一种调节机制，通过改变受体：g蛋白相互作用的有效性和特异性来调节细胞对细胞外信号的反应。最近的研究发现，在被诊断患有与自杀相关的焦虑、精神分裂症和抑郁症的患者中，以及在抗抑郁和抗精神病药物治疗后，5HT2C的表达发生了变化。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，我们已经证明了5HT2C mRNA和蛋白质异构体之间的差异，因为仅表达完全编辑的5HT2C受体异构体的转基因小鼠与野生型动物相比，5HT2C受体密度增加了前所未有的40至70倍，但5HT2C mRNA水平保持不变。因此，RNA编辑通过未知的转录后机制对5HT2C蛋白的表达产生显著影响。本提案的目标是开发新的转基因工具，通过产生含有盒式受体（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，其中突变可以通过重组酶介导的盒式交换引入，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，并且比同源重组更有效。在这里，我们将重点放在将表位标签引入内源性5HT2C位点上，从而更有效地纯化5HT2C受体蛋白，以便随后比较离散脑区域的mRNA和蛋白质异构体分布。通过检测受体表达、配体亲和力和信号传导的潜在改变，将在转染的异源细胞系中评估表位插入受体多个位点的功能后果。这个

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.