BHLH FACTOR IN DIABETES & PANCREAS DEVELOPMENT

糖尿病中的 BHLH 因素

• 批准号: 6711689
• 负责人: DEBRA E BRAMBLETT
• 金额: $ 8.99万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-15 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6711689
• 关键词: biological models; diabetes mellitus; disease /disorder model; gene expression; gene targeting; genetic promoter element; genetically modified animals; histogenesis; immunocytochemistry; in situ hybridization; laboratory mouse; molecular pathology; pancreas; protein binding; protein structure function; transcription factor

My career goal is to identify molecular events that govern pancreas development and functionality and the genetic components that delineate these events. Ultimately, such findings will contribute to better treatments and prevention of pancreatic disease. My current focus is to dissect the role of basic Helix-Loop-Helix (bHLH) factors in pancreatic endocrine cell development. I am currently characterizing a novel bHLH factor, BETA4, which is expressed during ontogeny of the pancreas. I plan to assess the molecular function of this factor in transcription of pancreatic genes. This will be accomplished via transient transfection assays in mammalian cells in culture. Once target promoters are identified, l will examine the ability of BETA4 to bind to DNA at specific sequences via gel retardation assays and to regulate transcription through promoter deletion analysis. However, to determine the physiological role of this factor, l am generating a loss-of- function mouse mutant. The BETA4 gene will be targeted for disruption in embryonic stem cells. In combination, the beta-galactosidase gene will be "knocked-in" such that it is a direct reflection of BETA4 expression. For this purpose, a targeting construct, 'which contains fragments of genomic DNA derived from the BETA4 locus flanking the beta-galactosidase gene and a selectable antibiotic marker gene, is being engineered. Analysis of the BETA4 knockout mouse will reveal both the precise location of BETA4 expression at the cellular level and the role BETA4 plays in the development or the functionality of the pancreas. The BETA4 knockout will be a mouse model system for the study of aspects of pancreas development and possibly pancreatic disease.

我的职业目标是确定控制胰腺发育和功能的分子事件以及描述这些事件的遗传成分。最终，这些发现将有助于更好地治疗和预防胰腺疾病。我目前的重点是剖析基本螺旋环阻碍（bHLH）因素在胰腺内分泌细胞发育中的作用。目前，我正在研究一种新的bHLH因子BETA4，它在胰腺个体发育过程中表达。我计划评估这个因子在胰腺基因转录中的分子功能。这将通过培养的哺乳动物细胞中的瞬时转染测定来完成。一旦鉴定出靶启动子，我将通过凝胶阻滞试验检测BETA 4在特定序列处结合DNA的能力，并通过启动子缺失分析调节转录。然而，为了确定这个因子的生理作用，我正在制造一个功能丧失的突变小鼠。BETA4基因将在胚胎干细胞中被靶向破坏。在组合中，β-半乳糖苷酶基因将被“敲入”，使得其直接反映BETA 4表达。为此目的，正在对靶向构建体进行工程改造，所述靶向构建体含有源自β-半乳糖苷酶基因侧翼的BETA 4基因座的基因组DNA片段和选择性抗生素标记基因。对BETA4基因敲除小鼠的分析将揭示BETA4在细胞水平表达的精确位置以及BETA4在胰腺发育或功能中的作用。BETA4基因敲除将成为研究胰腺发育和可能的胰腺疾病方面的小鼠模型系统。