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眼透镜生命中的生理和/或遗传易感性时期。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）最能证明这一点。这种发育异常最近已与热休克转录因子4（HSF 4）的DNA结合域中的突变有关。我们的研究表明，HSF 4出现在透镜的初始发育完成后，并在出生后的透镜中表达增加。这种表达模式提示与人透镜中层状白内障的出现有显著的相关性。为了了解控制基因型和病理表型之间的这种联系的分子机制，我们提出了3个特定的目标（SA）采用BAC（细菌人工染色体）转基因，这是基于最近的革命“工程”技术的BAC的操作。在第一个具体的目标，我们建议划定边界的转录单位的HSF 4基因，其启动子（S）的特点和阐明的复杂性，不同的异构体产生的选择性剪接。在第二个SA中，我们将把报告基因（LacZ或EGFP）工程化到位于BAC中的HSF 4基因中，制备转基因小鼠并跟踪报告基因的表达。由于BAC在空间和时间上指导正确表达，我们将能够精确定位HSF 4基因中和周围的必要DMA区域，这些区域是在眼透镜中忠实表达所需的。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4种突变（在人HSF 4基因中报道的）引入转基因小鼠中来创建人白内障发生的小鼠范例。我们将研究这些小鼠，以发现基因型的质量如何与眼透镜的形态学和分子表型的时间呈现相关，从而使我们深入了解白内障发生和HSF 4基因活性。

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.