TARGET GENE IDENTIFICATION OF EMX TRANSCRIPTION FACTORS

EMX转录因子的靶基因鉴定

• 批准号: 2546313
• 负责人: JAMES J CONTOS
• 金额: $ 1.45万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-10-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2546313
• 关键词: cerebral cortex; developmental genetics; developmental neurobiology; gene targeting; in situ hybridization; laboratory mouse; northern blottings; nucleic acid sequence; polymerase chain reaction; tissue /cell culture; transcription factor

The long term objectives of the research proposed is to understand the molecular and cellular basis for the development of the cerebral cortex. The specific hypothesis we aim to investigate is that the embryonic cerebral cortex-restricted transcription factors Emx1 and/or Emx2 regulate the transcription of target genes with limits of expression demarcating the developing cerebral cortex. The approach taken is to determine endogenous expression levels of Emx1 and Emx2 in various murine neural cell lines, including several derived from the cortex, using Northern blots. Choosing a cell line with little endogenous Emx expression, an inducible Emx1 and/or Emx2 gene will be stably integrated using a calcium phosphate transfection protocol. The Emx genes will be highly expressed after removal of low levels of tetracycline from the cell culture medium. mRNA transcripts that are up- or downregulated after Einx induction will be identified using RDA and PCR differential display. Subtraction products will be screened for embryonic cerebral cortex expression using in situ hybridization, and the most interesting of these will be characterized by DNA sequencing. Understanding the molecular interactions responsible for normal development of the cerebral cortex could shed light on the multitude of characterized problems with this organ, ranging from dyslexia to schizophrenia.

建议的研究的长期目标是了解 大脑皮层发育的分子和细胞基础。 我们要研究的具体假设是， 大脑皮层限制性转录因子Emx 1和/或Emx 2 调节靶基因的转录，限制表达 划分发育中的大脑皮质。所采取的方法是 测定各种组织中Emx 1和Emx 2的内源性表达水平， 鼠神经细胞系，包括几种来源于皮层的细胞系， 用北方印迹法。选择几乎没有内源性Emx的细胞系 表达时，可诱导的Emx1和/或Emx2基因将被稳定表达。 使用磷酸钙转染方案整合。Emx 基因将在去除低水平的 细胞培养基中的四环素。mRNA转录本- 将使用RDA鉴定Einx诱导后表达或下调的细胞， PCR差异显示。将筛选减影产品， 使用原位杂交的胚胎大脑皮层表达，和 其中最令人感兴趣的将通过DNA测序来表征。 了解分子间的相互作用， 大脑皮层的发育可以揭示 这个器官的典型问题，从阅读障碍到 精神分裂症