Estrogen Receptor Gene Transfer in Mouse Brains

描述(申请人提供)：这项建议的研究目标是测试使用腺相关病毒(AAV)的脑部位特异性基因转导是否能够在敲除小鼠的大脑中局部恢复被破坏的基因的表达和功能。我们一直在研究两种类型的雌激素受体(ER)在神经内分泌和行为功能的雌激素样调节中的不同作用，使用ER-α(αRKO)和ER-β(βERKO)基因敲除的小鼠。基因敲除小鼠是研究许多特征中特定基因功能并将它们全部关联起来的很好的工具，但有一个局限性，即目标基因被全局和永久地干扰。在这里，我们建议根据特定研究的终点，在特定大脑区域将被破坏的基因重新引入基因敲除小鼠。我们的初步研究表明，我们可以定点地将被破坏的基因ER-α或ER-β重新引入各自的基因敲除小鼠的大脑。还发现转导的ER基因是有功能的，因为孕激素受体蛋白是雌激素在转导细胞中诱导的，孕激素受体蛋白是ER配体依赖转录活性的下游产物。在拟议的研究中，我们将测试位点特异性AAV介导的基因转移在成年基因敲除小鼠大脑中的功能后果。具体地说，我们将通过将含有ER-beta基因的AAV载体(AAV.ER-beta)注射到BetaERKO雄性小鼠的下丘脑室旁核中，重新引入ER-6基因，并确定中断的神经内分泌和行为功能是否可以恢复。在拟议的研究的第一部分，将用原位杂交和定量聚合酶链式反应检测雌激素诱导的催产素基因表达上调的影响(目标I)。如果结果是肯定的，ER-13基因转导的行为效应将进一步从ER-β介导的雌激素的抗焦虑作用以及雌激素诱导的雄性BetaERKO小鼠的攻击行为(AIM II)方面进行检验。拟议的研究结果将提供关于两种类型内质网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.