ANALYSIS OF A MURINE HOMEO BOX LOCUS

这项研究计划解决了遗传控制的问题， 使用实验室小鼠作为模型系统在哺乳动物中的发育。 它 将利用最近在分析 发育控制基因形成果蝇，这导致了分离， 一种交叉同源的蛋白质编码DNA序列，称为同源盒。 的 事实上，这种高度保守的序列到目前为止只被发现 在果蝇基因发育控制功能， 证明它在其他动物的基因组中广泛存在 包括高等脊椎动物在内的群体，导致人们猜测， 同源框序列可以作为分子探针用于鉴定 哺乳动物也有发育控制基因。 假设这个假设 是正确的，含有基因家族的小鼠同源盒可以作为一种 研究哺乳动物遗传控制机制的模型系统 发展 拟议研究的长期目标是 表征先前分离的成员的结构和功能， 基因家族，命名为Hox 3.1。 由于同源异型盒基因被认为控制着靶基因的表达 通过反式调节机制，一组实验将集中在 通过蛋白质/DNA结合分离Hox 3.1特异性靶序列 研究将使用基因表达中产生的Hox 3.1蛋白 向量系统 此外，针对这种蛋白质产生的抗体将被 用于定位组织切片中的Hox 3.1蛋白。 第二种方法 Hox 3.1特异性靶基因的分离将基于 来自Hox 3.1表达的cDNA的消减杂交， 非表达培养细胞，携带诱导型Hox 3.1基因构建体。 Hox 3.1的发育控制功能的更直接证据可能 来自于在多能细胞中使用定点诱变的实验， 胚胎干细胞的目的是通过囊胚生成嵌合小鼠 注射 这些小鼠可能通过它们的基因传递突变的Hox 3.1基因。 生殖系，导致杂合子后代，然后可用于 通过适当的育种进一步分析可能的发育缺陷。 一 第二组生殖细胞转化实验旨在 介导区域特异性的顺式调节元件的鉴定 Hox 3.1的表达。 一系列DNA构建体， Hox 3.1 5'调控区的缺失片段， 报告基因将用于产生转基因小鼠或小鼠 胚胎 报告基因表达模式的差异可能 然后与确定的5' DNA片段的存在相关联。

This research proposal addresses the problem of genetic control of development in mammals using the laboratory mouse as a model system. It will take advantage of recent progress made in the analysis of developmental control genes form Drosophila, which lead to the isolation of a cross homologous, protein coding DNA sequence termed as homeo box. The fact that this highly conserved sequence has so far been found exclusively in Drosophila genes with developmental control functions, and the demonstration of its widespread occurrence in the genomes of other animal groups including higher vertebrates, lead to the speculation that the homeobox sequence may serve as a molecular probe for the identification of developmental control genes also in mammals. Provided that this assumption is correct, the murine homeo box containing gene family may serve as a model system for studying genetic control mechanisms of mammalian development. The long term objective of the proposed research is to characterize structure and function of a previously isolated member of this gene family, designated as Hox 3.1. Since homeobox genes are believed to control the expression of target genes via trans regulatory mechanisms, one set of experiments will focus on the isolation of Hox 3.1 specific target sequences by protein/DNA binding studies, which will employ Hox 3.1 protein produced in a gene expression vector system. In addition, antibodies raised against this protein will be used for localizing Hox 3.1 protein in tissue sections. A second approach for the isolation of Hox 3.1 specific target genes will be based on subtraction hybridization of cDNAs derived from Hox 3.1 expressing and nonexpressing culture cells, carrying an inducible Hox 3.1 gene construct. More direct evidence for a developmental control function of Hox 3.1 may come from experiments using site directed mutagenesis in pluripotent embryonic stem cells with the goal to generate chimeric mice by blastocyst injection. These mice may transmit the mutated Hox 3.1 gene through their germ line, leading to heterozygous offspring that may then be used for further analysis of possible developmental defects by proper breeding. A second set of germ cell transformation experiments aims at the identification of cis regulatory elements mediating region specific expression of Hox 3.1. A series of DNA constructs containing progressively deleted segments of the putative Hox 3.1 5' regulatory region linked to a reporter gene will be used for the generation of transgenic mice or mouse embryos. Differences in the expression patterns of the reporter gene may then be correlated with the presence of defined 5' DNA segments.