IN VIVO MUTAGENESIS STUDIES USING TRANSGENIC MICE

工作总结：当特定的细胞突变机制途径 通常是最方便的体外研究，组织的差异 反应，化学沉积或不同细胞的反应 诱变剂/致癌物暴露的类型只能在体内确定。大的 Blue携带30-40个拷贝的Lambda噬菌体，每个噬菌体带有一个LacL拷贝 它被用作突变目标。最近，lambda CLL基因 也被定义为有用的突变靶基因；因此提供了 用于体内诱变研究的LACL的替代品。而LACL有 作为突变研究的有用标记，CLL检测提供了 与LACL生成的数据类似的定量数据允许 以更少的操作和更低的成本进行研究 已生成数据点。随着转基因动物的发展，对 体内突变的研究，组织对一种 诱变剂/致癌物现在可以确定了。我们已经定义了 大蓝鼠lacL转基因的自发突变是 在选定的组织中确定。在所有组织中，占主导地位的一类 突变为G：C AE A：T转换，大部分发生在5‘-CpG-3’ 二核苷酸序列。第二种最常见的突变类型是G：C AET：一种颠倒。这些数据被用来建立一个数据库 在大蓝鼠身上自发产生的自发突变，因此 作为治疗后恢复的突变的参照的 可以与之相比。简而言之，治疗的时机和部分 在这些研究中，肝叶切除术被证明是至关重要的。我们已经研究过 细胞增殖对突变频率的影响 在雄性C57B1/6大蓝转基因小鼠的肝脏中产生 给小鼠进行肝部分切除，然后用 苯并(A)芘(Benzo(A)P)。我们还证明了致癌物质 阻燃剂三(2，3-二溴丙基)磷酸(TDBP)具有致突变性 肾脏，它是观察到肿瘤的同一组织 雄性和雌性大鼠以及雄性小鼠。我们打算继续研究 DNA代谢中的特定缺陷对血管紧张素转换酶活性的影响 体内诱变。具体地说，细胞形成lacl/polb基因敲除小鼠 可供研究。通过评估LACL或LACL中的突变 CLL基因，我们将深入了解b- 聚合酶在正常复制和修复复制中都存在。此外，我们还将 开始协调体外和体内的诱变研究 目前正在实验室中，专注于使用不匹配进行的研究 修复(MMR)缺陷转基因小鼠及其衍生的细胞系 老鼠。

SUMMARY OF WORK: While specific cellular mutational mechanistic pathways are often most conveniently studied in vitro, differences in tissue response, in chemical deposition or in the response of different cell types to mutagen/carcinogen exposure can only be defined in vivo. Big Blue carries 30-40 copies of lambda phage each with a single copy of lacl which is used as a mutational target. More recently, the lambda cll gene has also been defined as a useful mutational target gene; thus providing an alternative to lacl for in vivo mutagenesis studies. While lacl has been a useful marker for mutagenesis studies, the cll assay provides quantitative data similar to those generated with lacl, allows for studies to be performed with fewer manipulations and is less costly per data point generated. With the development transgenic animals useful for the study of in vivo mutations, the tissue specific response to a mutagen/carcinogen can now be determined. We have defined the nature of spontaneous mutations in the lacl transgene of Big Blue mice was determined in selected tissues. In all tissues, the predominant class of mutations was G:C AE A:T transitions, most of which occurred at 5'-CpG-3' dinucleotide sequences. The second most common class of mutations was G:C AE T:A transversions. These data have been used to establish a database of spontaneous mutations that arise spontaneously in Big Blue mice, thus serving as a reference against which mutations recovered after treatment can be compared. In short the timing of treatment and of partial hapatectomy were shown to be critical in these studies. We have studied the effects of cellular proliferation on the frequency of mutations arising in the liver of male C57B1/6 Big Blue transgenic mice by subjecting the mice to partial hepatectomy following treatment with benzo(a)pyrene (B[a]P). We have also demonstrated that the carcinogenic flame retardant tris(2,3-dibromopropyl)phosphate (TDBP) is mutagenic in the kidney which is the same tissue for which tumors were observed in male and female rats and in male mice. We intend to continue to study the effects of specific defects in DNA metabolism on the process of mutagenesis in vivo. Specifically, cells form lacl/pol b -knockout mice are available for study. By evaluating mutations in either the lacl or the cll genes, we will gain insight regarding the role of the b- polymerase in both normal and repair replication. Furthermore, we will begin to coordinate the in vitro and in vivo mutagenesis studies that are presently on-going in the lab by focusing on studies using mismatch repair (MMR) defective transgenic mice and cell lines derived from these mice.