ANALYSIS OF A MURINE HOMEO BOX LOCUS

鼠同源框基因座的分析

基本信息

批准号：
3467958
负责人：
ALEXANDER AWGULEWITSCH
金额：
$ 9.6万
依托单位：
MEDICAL UNIVERSITY OF SOUTH CAROLINA
依托单位国家：
美国
项目类别：
财政年份：
1989
资助国家：
美国
起止时间：
1989-04-01 至 1994-09-11
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3467958
关键词：
DNA binding protein developmental genetics embryonic stem cell gene expression genetic library genetic promoter element genetic regulatory element homeobox genes immunocytochemistry laboratory mouse laboratory rabbit molecular cloning nucleic acid sequence site directed mutagenesis subtraction hybridization tissue /cell culture transfection

项目摘要

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.

该研究计划解决了基因控制问题使用实验室小鼠作为模型系统在哺乳动物中进行发育。它将利用最近在分析方面取得的进展果蝇的发育控制基因，导致分离交叉同源的蛋白质编码 DNA 序列，称为同源盒。这事实上，这个高度保守的序列迄今为止仅被发现果蝇基因中具有发育控制功能，证明其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，导致人们猜测同源盒序列可以作为分子探针来鉴定发育控制基因也存在于哺乳动物中。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该先前孤立成员的结构和功能基因家族，指定为Hox 3.1。由于同源盒基因被认为控制靶基因的表达通过反式监管机制，一组实验将集中于通过蛋白质/DNA 结合分离 Hox 3.1 特定靶序列研究将利用基因表达中产生的 Hox 3.1 蛋白矢量系统。此外，针对该蛋白质产生的抗体将用于定位组织切片中的 Hox 3.1 蛋白。第二种方法用于分离 Hox 3.1 的特定靶基因将基于源自表达 Hox 3.1 的 cDNA 的消减杂交非表达培养细胞，携带可诱导的 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使用多能定点诱变的实验胚胎干细胞的目标是通过囊胚产生嵌合小鼠注射。这些小鼠可能通过其身体传播突变的 Hox 3.1 基因种系，产生杂合后代，然后可用于通过适当的育种进一步分析可能的发育缺陷。一个第二组生殖细胞转化实验旨在鉴定介导区域特异性的顺式调控元件 Hox 3.1 的表达。一系列 DNA 构建体逐渐包含删除了假定的 Hox 3.1 5' 监管区域的片段，链接到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。