Gene Expression in Normal and Cataractous Lens

描述：儿童早期和老年可能仅仅代表了晶状体生命中生理和/或遗传易感性的时期。板层/马南白内障（常染色体显性）出现在儿童早期或青少年时期最好的例子。这种发育异常最近被认为与热休克转录因子4 (HSF4) DNA结合域的突变有关。我们的研究表明，HSF4在晶状体发育完成后出现，并在出生后晶状体中表达增加。这种表达模式提示与人类晶状体板层性白内障的出现有显著的联系。为了了解控制基因型和病理表型之间这种联系的分子机制，我们提出了三个特定目的（SA），利用BAC（细菌人工染色体）转基因，这是基于最近的“重组”技术革命来操纵BAC。在第一个具体目标中，我们建议划定HSF4基因转录单元的边界，表征其启动子，并阐明由选择性剪接产生的不同同种异构体的复杂性。在第二种方法中，我们将在BACs中的HSF4基因中植入一个报告基因（LacZ或EGFP），制作转基因小鼠并追踪报告基因的表达。由于BACs在空间和时间上指导正确的表达，我们将能够确定HSF4基因内和周围的必需DMA区域，这些区域是晶状体中忠实表达所必需的。使用在特定目标#2中鉴定的BAG，在SA #3中，我们将通过将相同的四种突变（在人类HSF4基因中报道）引入转基因小鼠中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，以发现基因型的质量如何与晶状体的形态和分子表型的时间表现相关，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: 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 three 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 four 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.