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 将弯曲尾（Cd）小鼠中突变的基因表征为新的位点 与叶酸（FA）敏感性神经管缺陷（NTD）相关。纯合 cd倾向于吻侧NTD，完成神经胚形成的人显示出微妙的 皮质发育不良在前三年的资助中，我们已经证明， 膳食叶酸可降低镉引起的NTD发生率， 类似于临床观察，使其成为人NTD的重要模型。 连锁分析将Cd基因定位在染色体的0.2cM区域 6. Cd关键区域的物理制图和排序已经确定了3个 候选基因该项目将确定镉基因，调查 其CNS畸形在细胞和分子水平上的发病机制， 检查叶酸代谢和这些缺陷之间的关系。第一，CD 基因将通过定位克隆和鉴定的测试来寻找 候选人连锁分析已完成，基因组DNA重叠群 覆盖关键区域。在更新期间，我们 将通过以下方式鉴定Cd：（a）克隆与基因组重叠群对应的cDNA;（B） 分析来自Cd中大突变和小突变区域的候选基因 小鼠产生镉的基因的身份将通过 证明可以通过引入BAC来拯救表型 包括整个候选基因。或者，推定的Cd突变 将被"敲入"以重现Cd表型。第二，机制 将确定是否由于改变的细胞 增殖、神经元迁移或程序性细胞死亡。形态发生 将通过延时共聚焦显微镜检查培养中的单个镉胚胎。 显微镜此外，将使用以下标记物定义Cd脑组织发生： 神经命运确定和CNS模式形成。第三，调查 饮食叶酸将继续，以确定是否FA改变增殖 以及FA是否也能改善神经胚形成过程中细胞的Cd 皮质发育不良第四，镉的功能研究将开始， 基因产物的结构分析。FA相关代谢状况 镉动物的途径将被调查的线索，潜在的机制 导致FA敏感的NTD。功能将通过过度表达进行研究 的镉在小鼠中，由BAC载体引入，以允许转基因表达， 适当的时间和解剖顺序。进一步的功能研究将 通过条件同源重组基因敲除在正常小鼠中抑制Cd。 研究导致遗传异常的遗传、分子和细胞事件， Cd将有助于对NTD的机理理解，并可能导致 对单个家庭进行产前评估的策略？的风险和量身定制 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.