GENETICS OF NOTOCHORD DEVELOPMENT

描述：（改编自研究者摘要）这是第一次 要求五年支持的RO 1应用程序的竞争性续订 通过鉴定Sd中突变的基因来研究脊索发育， 进入点自首次颁发补助金以来的三年里， 申请人使用胚胎嵌合体研究来确定Sd和tc是否 （另一种影响脊索发育的突变）在细胞中自主作用 的方式，并启动了一系列的实验，以分离Sd的基础上， 地图位置本申请提出完成这些实验 并开始在基因水平上表征Sd基因产物， 蛋白质表达目前，Sd局部化到0.3 cM间隔;一侧 用650 kb BAC重叠群（其中600 kb 是非重组的），另一侧由分子标记D2 Uc 13限定， 与Sd的距离为7/2919。在第一个具体目标中， 申请人将使用BAC通过染色体步移完成物理图谱， YACs，并尝试通过BAC转基因缩小临界区域 拯救实验，并通过在新的研究中产生额外的重组动物， 回交(The申请人在补助金审查之前提供了最新情况， 额外的两个染色体步移步骤已经关闭了物理间隙， 将临界区域缩小到<750 kb）。第二个具体目标是 在Sd的分子鉴定中，将通过以下步骤搜索候选基因： 如果关键区域可以缩小到 <150 kb;如果该区域更大，将使用 E9.5-10.5全胚组织。候选基因的初步验证 因为Sd中的突变将基于表达和序列结构分析 比较突变和正常组织，然后用转基因方法， 拯救或繁殖突变表型。最后，在第三个具体目标中， Sd基因产物的机制研究将通过分离 全长cDNA，产生抗体，并进行详细的分析 RNA和蛋白质的表达。

DESCRIPTION: (Adapted from investigator's abstract) This is the first competitive renewal of an RO1 application that requests five years of support to study notochord development by identifying the gene mutated in Sd as an entry point. In the three years since the grant was first awarded, the applicant used embryonic chimera studies to determine whether or not Sd and tc (another mutation that affects notochord development) act in a cell-autonomous manner, and initiated a series of experiments to isolate Sd on the basis of its map position. The current application proposes to complete these experiments and to begin characterizing the Sd gene product at the level of gene and protein expression. At present, Sd is localized to a 0.3 cM interval; one side of the interval is closed physically with a 650 kb BAC contig (of which 600 kb is non-recombinant), the other side is defined by a molecular marker, D2Ucl3, that lies 7/2919 recombinants away from Sd. In the first specific aim the applicant will complete the physical map by chromosome walking using BACs and YACs, and try to narrow the critical region by carrying out BAC transgenic rescue experiments and by generating additional recombinant animals in a new backcross. (The applicant provided an update prior to the grant review that an additional two steps of chromosome walking had closed the physical gap, narrowing the critical region to <750 kb). In the second specific aim directed at molecular identification of Sd, a search for candidate genes will proceed by sequence sampling of 1-2 BAC clones if the critical region can be narrowed to <150 kb; if the region is larger, cDNA selection will be carried out using tissue from E9.5-10.5 whole embryos. Initial verification of a candidate gene as mutated in Sd will be based on expression and sequence structure analyses that compare mutant and normal tissues, followed by transgenic approaches to rescue or reproduce the mutant phenotype. Finally, in a third specific aim, mechanistic studies of the Sd gene product will be initiated by isolating a full-length cDNA, generating antibodies, and carrying out a detailed analysis of RNA and protein expression.