Crooked Tail: Gene Expression in a Neural Tube Defect

描述(由申请人提供)：这个正在进行的项目的目标是 扭曲尾巴(CD)小鼠突变基因的新基因特征 与叶酸(FA)敏感型神经管缺陷(NTD)相关。纯合子 CD容易出现吻部NTD，而完成神经支配的人表现出微妙的 皮质发育不良。在前3年的资助中，我们已经证明了 在CD中，饮食中叶酸以一种密切的方式减少NTD的发生率 类似于临床观察，使其成为人类NTD的重要模型。 连锁分析已将CD基因座精细定位到0.2 cM的染色体区域 6.CD关键区域的物理测绘和排序已确定3 候选基因。该项目将鉴定CD基因，调查 其中枢神经系统畸形的细胞和分子水平及发病机制 检查叶酸代谢与这些缺陷之间的关系。第一，CD 将通过定位克隆和检测已识别的基因来寻找基因 候选人。连锁分析已经完成，基因组DNA重叠群 已经建立了覆盖关键区域的机制。在续约期内，我们 将通过以下方式识别CD：(A)克隆与基因组重叠群对应的cDNA；(B) CD大、小突变区域候选基因分析 老鼠。产生CD的基因的身份将通过 证明通过引入BAC可以挽救表型 包括整个候选基因。或者，假定的CD突变 将被“敲进去”来概括CD的表型。第二，机制 导致CD表型的原因将被确定，是否由于细胞改变 增殖、神经元迁移或程序性细胞死亡。形态发生学 培养中的单个CD胚胎将通过时间推移共聚焦进行检测 显微镜。此外，CD脑组织发生将使用以下标记来定义 神经命运决定和中枢神经系统模式的形成。第三，调查 饮食中的叶酸将继续，以确定FA是否改变增殖 以及FA是否也能改善CD的脑组织 皮质发育不良。第四，CD的功能研究将从 基因产物的结构分析。脂肪酸相关代谢研究现状 CD动物的通路将被研究，以寻找潜在机制的线索 导致对FA敏感的NTD。函数将通过过度表达进行调查 Cd在小鼠体内的表达，由BAC载体引入，以允许转基因在 适当的时间和解剖顺序。进一步的功能研究将 通过有条件的同源重组敲除使正常小鼠的CD失活。 遗传、分子和细胞事件导致的异常的研究 CD将有助于对NTD的机械性理解，并可能导致 个别家庭产前评估的策略--S风险与量身定制 预防人脑发育不良。

DESCRIPTION (provided by applicant): The goal of this ongoing project is to characterize the gene mutated in the Crooked tail (Cd) mouse as a new locus associated with folic acid (FA) sensitive neural tube defects (NTD). Homozygous Cd are prone to rostral NTD and those completing neurulation display a subtle cortical dysplasia. In the first 3 years of funding, we have shown that the incidence of NTD is reduced in Cd by dietary folate in a manner closely resembling clinical observation, making it an important model for human NTD. Linkage analysis has fine-mapped the Cd locus to a 0.2 cM region of chromosome 6. Physical mapping and sequencing of the Cd critical region has identified 3 candidate genes. The project will identify the Cd gene, investigate the pathogenesis of its CNS malformations at the cellular and molecular levels and examine the relation between folate metabolism and these defects. First, the Cd gene will be sought through positional cloning and testing of identified candidates. Linkage analysis has beer completed and a genomic DNA contig covering the critical region has been established. In the renewal period we will identify Cd by: (a) cloning cDNAs corresponding to the genomic contig; (b) analysis of candidate genes from the region for large and small mutations in Cd mice. The identity of the gene producing Cd will be confirmed through demonstration that the phenotype can be rescued by introduction of BACs encompassing at entire candidate gene. Alternatively, the putative Cd mutation will be "knocked-in" to recapitulate the Cd phenotype. Second, The mechanisms leading to the Cd phenotype will be determined, whether due to altered cell proliferation, neuronal migration or programmed cell death. Morphogenesis of individual Cd embryos in culture will be examined by time-lapse confocal microscopy. In addition Cd brain histogenesis will be defined using markers of neural fate determination and CNS pattern formation. Third, Investigation of dietary folate will continue, to determine whether FA alters the proliferation of cells during neurulation, and whether FA can also ameliorate Cd cerebral cortical maldevelopment. Fourth, Functional studies of Cd will begin with structural analysis of the gene product. The status of FA-related metabolic pathways in Cd animals will be investigated for clues to potential mechanisms leading to FA-sensitive NTD. Function will be investigated by over-expression of Cd in mice, introduced by BAC vectors to permit transgene expression in appropriate temporal and anatomic sequence. Further functional studies will inactivate Cd in normal mice by a conditional homologous recombinant knockout. Study of the genetic, molecular and cellular events leading to abnormalities in Cd will contribute to mechanistic understanding of NTD and may lead to strategies for prenatal assessment of an individual family?s risk and tailored prevention of human brain maldevelopment.