BHLH FACTOR IN DIABETES & PANCREAS DEVELOPMENT

糖尿病中的 BHLH 因素

• 批准号: 6380183
• 负责人: DEBRA E BRAMBLETT
• 金额: $ 8.09万
• 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 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6380183
• 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-螺旋(BHLH)因子在胰腺内分泌细胞发育中的作用。我目前正在研究一种新的bHLH因子，Beta4，它在胰腺的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。这将通过在培养的哺乳动物细胞中进行瞬时转基因分析来实现。一旦确定了目标启动子，L将通过凝胶滞留分析来检测Beta4与特定序列上的DNA结合的能力，并通过启动子缺失分析来调节转录。然而，为了确定这一因素的生理作用，L正在产生一个功能丧失的小鼠突变体。Beta4基因将成为破坏胚胎干细胞的目标。结合起来，β-半乳糖苷酶基因将被“敲入”，从而直接反映Beta4的表达。为此，正在设计一种靶向结构，它包含来自β-半乳糖苷酶基因两侧的Beta4基因座的基因组DNA片段和一个可选择的抗生素标记基因。对Beta4基因敲除小鼠的分析将揭示Beta4基因在细胞水平上的准确表达位置以及Beta4在胰腺发育或功能中所起的作用。Beta4基因敲除将成为研究胰腺发育和可能的胰腺疾病的小鼠模型系统。