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GENE TRANSFER AND GENE EXPRESSION IN MAMMALIAN CELLS

哺乳动物细胞中的基因转移和基因表达

基本信息

批准号：
3276350
负责人：
GEORGE A SCANGOS
金额：
$ 9.62万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1983
资助国家：
美国
起止时间：
1983-02-01 至 1986-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3276350
关键词：
Alphaherpesvirinae gene expression genetic manipulation molecular cloning nucleic acid sequence thymidine kinase tissue /cell culture tissue mosaicism

项目摘要

Gene transfer is a powerful tool for the analysis of factors which control gene expression in mammalian cells. We have transferred the Herpes simplex virus thymidine kinase (TK) gene into mouse LTK cells and have obtained cell lines which physically retain the genes but modulate expression when selection against TK is applied. The efficient selections for and against expression of TK coupled with the use of the cloned viral gene as a nucleic acid probe provide a powerful system with which to correlate changes in DNA and/or chromatin structure with gene expression. We propose to analyze several cell lines, some of which switch from TK+ to TK- and back at a frequency of 10 to the -6, and others which undergo the same phenotypic changes at a frequency of 10 to the -2, for changes in DNA which correlate with and cause changes in gene expression. In a second set of experiments, the normal 5' flanking DNA (promoter region) of the TK gene has been replaced with randomly cloned mouse DNA, and several sequences which restore the ability of the gene to function in gene transfer experiments have been identified. Such sequences might be normal cellular promoters, origins of DNA replication, or enhancers of transcription and/or integration. We propose to isolate and characterize several such sequences to elucidate features which are important for gene expression. The final set of experiments will use gene transfer experiments to determine if exogenous DNA can be directed to certain areas of the genome and if a mutant cellular gene can be resolved by recombination with an incoming gene. These experiments may point out ways to perform gene transfer experiments to ask more sophisticated experiments than currently possible.

基因转移是分析控制基因的因素的有力工具。哺乳动物细胞中的基因表达。我们已经将单纯疱疹病毒病毒胸苷激酶(TK)基因导入小鼠LTK细胞并获得细胞系物理上保留基因，但在应用针对TK的选择。支持和反对的有效选择 TK的表达及其与克隆病毒基因的结合酸性探针提供了用于关联DNA变化的强大系统和/或染色质结构与基因表达。我们建议分析几个细胞系，其中一些在TK+和TK-之间切换频率为10到-6，以及其他具有相同表型的频率为10到-2的变化，用于相关的DNA变化引起基因表达的变化。在第二组实验中， TK基因正常的5‘侧翼DNA(启动子区域)已经被取而代之的是随机克隆的小鼠DNA和几个序列在基因转移实验中恢复基因的功能已经被确认了。这样的序列可能是正常的细胞启动子， DNA复制的起源，或转录和/或增强剂整合。我们建议分离和表征几个这样的序列阐明对基因表达具有重要意义的特征。决赛一系列实验将使用基因转移实验来确定外源DNA可以被定向到基因组的某些区域，如果突变的细胞基因可以通过与传入的吉恩。这些实验可能会指出进行基因转移的方法。实验要求进行比目前可能的更复杂的实验。