ANALYSIS OF A MURINE HOMEO BOX LOCUS

这项研究建议解决的是遗传控制的问题。 以实验小鼠为模型系统在哺乳动物中的发育。它 将利用最近在分析方面取得的进展 来自果蝇的发育控制基因，导致分离出 一种交叉同源的蛋白质编码DNA序列，称为Homeo box。这个 到目前为止，这个高度保守的序列只被发现 在具有发育控制功能的果蝇基因中， 证明它广泛存在于其他动物的基因组中 包括高等脊椎动物在内的群体，导致了人们的猜测 同源框序列可作为分子探针用于鉴定 发育控制基因在哺乳动物中也存在。假设这一假设 是正确的，包含基因家族的小鼠同源盒可能作为一个 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 描述以前被隔离的这一部件的结构和功能 基因家族，命名为Hox 3.1。 由于同源框基因被认为控制着靶基因的表达 通过跨调控机制，一组实验将集中在 蛋白质/DNA结合法分离HOX 3.1特异性靶序列 研究，将使用在基因表达中产生的HOX 3.1蛋白 向量系统。此外，针对这种蛋白质产生的抗体将是 用于在组织切片中定位HOX 3.1蛋白。第二种方法 对于HOX 3.1的分离，特定的靶基因将基于 表达和表达HOX 3.1基因的cDNA的消减杂交 不表达的培养细胞，携带可诱导的HOX 3.1基因构建。 关于HOX 3.1发育控制功能的更直接证据可能 来自在多能性中使用定点突变的实验 胚胎干细胞，目标是通过囊胚产生嵌合体小鼠 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 通过适当的选育进一步分析可能的发育缺陷。一个 第二套生殖细胞转化实验旨在 识别调节区域特异性的顺式调节元件 HOX 3.1的表达。一系列DNA构造物，其中包含逐步 删除了推测的HOX 3.1 5‘调控区域片段，该区域与 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 然后与定义的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.