Gene Expression in Normal and Cataractous Lens

描述(由申请人提供)：儿童早期和老年可能仅仅代表了人工晶状体生命中生理和/或遗传易感性的时期。出现在儿童早期或青少年时期的板层/Marner白内障(常染色体显性遗传)就是最好的例证。这种发育异常最近被认为与热休克转录因子4(HSF4)的DNA结合域的突变有关。我们的研究表明，HSF4在晶状体的初始发育完成后出现，并在出生后的晶状体中表达增加。这种表达模式表明，与人类晶状体中板层白内障的出现有显著的关联。为了了解基因和病理表型之间的这种联系的分子机制，我们提出了利用细菌人工染色体(BAC)转基因的3个特异性目标(SA)，这是基于最近用于操纵BAC的“重组工程”技术的革命。在第一个特定目标中，我们建议划定hsf4基因转录单元的边界，表征其启动子(S)，并阐明通过选择性剪接产生的不同亚型的复杂性。在第二个SA中，我们将一个报告者(LacZ或EGFP)整合到BAC中的HSF4基因中，制造转基因小鼠，并跟踪报告者的表达。因为BAC引导正确的表达，在空间和时间上，我们将能够准确地定位HSF4基因及其周围的必要的DMA区域，这些区域是在眼晶状体中忠实表达所必需的。使用在特定目的#2中确定的袋子，在SA#3中，我们将通过将相同的4个突变(在人类HSF4基因中报告)引入转基因小鼠来创建人类白内障发生的小鼠范例。我们将对这些小鼠进行研究，以找出基因质量如何与眼晶状体的形态和分子表型的时间呈现有关，从而使我们能够深入了解白内障的发生和HSF4基因的活性。

DESCRIPTION (provided by applicant): Early childhood and old age may simply represent periods of physiological and/or genetic susceptibility in the life of an ocular lens. The lamellar/ Marner cataracts (autosomal dominant) that appear in the early childhood or juvenile years best exemplify this. This developmental aberration has been recently linked to mutations in the DNA binding domain of the heat shock transcription factor 4 (HSF4). Our investigations have revealed that HSF4 appears after the initial development of the lens is complete and increases in expression in the postnatal lens. This pattern of expression suggests a remarkable association with the appearance of the lamellar cataracts in the human lens. In order to understand the molecular mechanism that governs this link between the genotype and the pathological phenotype we propose 3 specific aims (SA) employing BAC (bacterial artificial chromosome) transgenesis that is based on recent revolution in "Recombineering" techniques for the manipulation of the BACs. In the first specific aim, we propose to delineate the boundaries of the transcriptional unit of the HSF4 gene, characterize its promoter(s) and elucidate the complexity of the different isoforms produced by alternative splicing. In the second SA, we will engineer a reporter (a LacZ or EGFP) into the HSF4 gene residing in BACs, make transgenic mice and follow the expression of the reporter. Because BACs direct correct expression, spatially and temporally, we will be able to pinpoint essential DMA regions in and around the HSF4 gene that are required for the faithful expression in the ocular lens. Using the BAG identified in specific aim #2, in the SA #3 we will create a mouse paradigm of human cataractogenesis by introduction of the same 4 mutations (reported in the human HSF4 gene) into transgenic mice. We will investigate these mice to find how the quality of the genotype relates to the temporal presentation of the morphological and the molecular phenotype of the ocular lens, allowing us an insight into cataractogenesis and HSF4 gene activity.