Estrogen Receptor Gene Transfer in Mouse Brains

描述（由申请人提供）：本提案的研究目的是测试使用腺相关病毒（AAV）进行脑位点特异性基因转导是否可以局部恢复敲除小鼠脑中被破坏基因的表达和功能。我们一直在研究两种类型的雌激素受体（ER），α和β，在神经内分泌和行为功能的雌激素调节中的不同作用，使用ER α（alphaRKO）和ER β（betaERKO）基因敲除小鼠。敲除小鼠是研究许多性状中特定基因功能并将其全部关联的重要工具，但其局限性在于靶基因被全局和永久破坏。在这里，我们建议根据特定研究的终点，在特定脑区重新引入被破坏的基因敲除小鼠。我们的初步研究表明，我们可以位点特异性地将被破坏的基因ER-α或ER-β重新引入各自的敲除小鼠大脑。还发现，转导的ER基因是功能性的，因为孕激素受体蛋白，ER的配体依赖性转录活性的最好表征的下游产物，在转导的细胞中被雌激素诱导。在拟议的研究中，我们将测试成年敲除小鼠脑中位点特异性AAV介导的基因转移的功能后果。具体而言，我们将通过将含有ER-β cDNA的AAV载体（AAV. ER-β）注射到betaERKO雄性小鼠的下丘脑室旁核中来重新引入ER-6基因，并确定是否可以恢复被破坏的神经内分泌和行为功能。在拟议的研究的第一部分，对雌激素诱导的催产素基因表达上调的影响将进行检查与原位杂交和定量PCR分析（目的I）。如果结果为阳性，将进一步研究ER-13基因转导对ER-β介导的雌激素抗焦虑作用以及雌激素诱导的雄性β ERKO小鼠攻击行为的影响（Aim II）。拟议的研究结果将提供有关两种类型的ER在大脑中的作用机制的有价值的信息。

DESCRIPTION (provided by applicant): The Research objective of this proposal is to test whether brain site-specific gene transduction with the use of adeno-associated virus (AAV) can focally restore expression and functions of a disrupted gene in knockout mouse brains. We have been investigating the differential roles of two types of estrogen receptors (ERs), alpha, and beta, in estrogenic regulation of neuroendocrine and behavioral functions, using knockout mice for ER-alpha (alphaRKO) and ER-beta (betaERKO) genes. Knockout mice are great tools to study specific gene function in many traits and correlate them all, but have the limitation that targeted genes are disrupted globally and permanently. Here, we propose to re-introduce a disrupted gene to knockout mice in a specific brain region depending on the endpoint of specific studies. Our preliminary studies demonstrated that we could site-specifically re-introduce a disrupted gene, ER-alpha or ER-beta, to respective knockout mouse brains. It was also found that transduced ER gene was functional, since progesterone receptor protein, the most well characterized down stream product of ligand-dependent transcriptional activity of ER, was induced by estrogen in the transduced cells. In the proposed studies, we will test the functional consequences of site-specific AAV mediated gene transfer in adult knockout mouse brains. Specifically, we will re-introduce ER-6 gene by injecting AAV vectors containing ER-beta Cdna (AAV.ER-beta) into the hypothalamic paraventricular nucleus of betaERKO male mice and determine whether disrupted neuroendocine and behavioral functions can be restored. In the first part of the proposed studies, the effects on estrogen-inducible up-regulation of oxytocin gene expression will be examined with in situ hybridization and quantitative PCR assays (Aim I). Given a positive outcome, behavioral effects of ER-13 gene transduction will be further examined in terms of ER-beta mediated anxiolytic action of estrogen as well as estrogen-inducible aggression in male betaERKO mice (Aim II). The outcome of the proposed studies will provide valuable information about mechanisms of action of two types of ERs in the brain.