DEVELOPMENTAL CONTROL OF INTRODUCED GENES IN RODENTS

我们建议研究特定DNA序列的作用，以及 这些序列在基因组中的位置在发育过程中 基因表达的调控。小鼠乳腺肿瘤基因片段的克隆 病毒(MTV)，其转录是由糖皮质激素诱导的，将是 通过显微注射将其导入小鼠受精卵。注射的胚胎将 被移植到养母体内，成年小鼠从这些 受精卵。然后这些小鼠将被培育成具有外源基因的后代 在不同的染色体上引入了MTV的拷贝。Southern印迹 分析将被用来识别含有新获得的 序列分析，同时对从猪组织中分离的RNA进行Northern杂交分析 这些小鼠将被用来确定哪些组织中的病毒序列 都被表达出来。这些外源引入的DNA将包含：序列 来自整个前病毒；含有基因缺失的前病毒 在体外进行工程；连接HSV-1胸苷的前病毒元件 或连接到小鼠和人c-myc基因的前病毒元件。 我们的目标是确定病毒序列和/或染色体 MTV在乳腺中的位置决定其受类固醇调节的表达 组织以及外源引入的序列是否会在 非乳腺组织。我们还将确定MTV序列是否可以 在荷尔蒙和发育调节中对非病毒DNA产生影响 时尚。因为MTV基因的表达是受荷尔蒙控制的 仅限于小鼠的哺乳期乳腺和肿瘤，该方法 所描述的内容将有助于理解 类固醇诱导基因调控的研究进展。

We propose to examine the role of specific DNA sequences, and the effect of the position of these sequences within the genome on the developmental regulation of gene expression. Cloned fragments of mouse mammary tumor virus (MTV), whose transcription is induced by glucocorticoids, will be introduced into mouse zygotes by microinjection. The injected embryos will be implanted into foster mothers, and adult mice derived from these zygotes. These mice will then be bred to give offspring with exogenously introduced copies of MTV at different chromosomal loci. Southern blot analysis will be used to identify mice that contain newly acquired sequences, while Northern blot analysis of RNA isolated from tissues of these mice will be used to determine in which tissues the viral sequences are expressed. These exogenously introduced DNAs will contain: sequences from the whole provirus; proviruses containing deletions genetically engineered in vitro; proviral elements to ligated the HSV-1 thymidine kinase gene; or proviral elements ligated to murine and human c-myc genes. Our goal is to determine whether the viral sequences and/or the chromosomal position of MTV determines its steroid-regulated expression in mammary tissue and whether an exogenously introduced sequence will be expressed in non-mammary tissue. We will also determine whether the MTV sequences can exert an effect on nonviral DNA in a hormonal and developmentally regulated fashion. Because MTV gene expression is hormonally controlled and is restricted to lactating mammary glands and tumors in mice, the approach described will lead to an understanding of the mechanisms involved in the development of regulation of a steroid inducible gene.