IN VIVO MUTAGENESIS STUDIES USING TRANSGENIC MICE

工作总结：虽然特定的细胞突变机制途径 通常最方便的是在体外进行研究， 反应，在化学沉积或在不同细胞的反应 诱变剂/致癌物暴露的类型只能在体内确定。大 蓝色携带30-40个λ噬菌体的拷贝，每个λ噬菌体都有一个LACL的拷贝 其被用作突变靶标。最近，λ 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的替代物。虽然lacl有 作为诱变研究的有用标记，CLL测定提供了 类似于用LACL产生的定量数据， 研究的操作较少，成本较低， 生成的数据点。随着转基因动物的发展， 体内突变的研究，组织特异性反应， 现在可以确定诱变剂/致癌物。我们已经定义了 大蓝小鼠的lacl转基因中的自发突变是 在选定的组织中测定。在所有组织中， 突变主要发生在5 '-CpG-3'端，G：CAEA：T转换， 二核苷酸序列。第二类最常见的突变是G：C AE T：A颠换。这些数据已被用于建立数据库 蓝色大老鼠的自发突变， 作为治疗后恢复突变的参照 可以比较。总之，治疗的时机和部分 在这些研究中，肝切除术被证明是关键。我们研究了 细胞增殖对突变频率的影响 在雄性C57 B1/6大蓝转基因小鼠的肝脏中产生， 在用以下处理后对小鼠进行部分肝切除术： 苯并（a）芘（B[a]P）。我们还证明了致癌物质 阻燃剂磷酸三（2，3-二溴丙基）酯（TDBP）在 肾脏是与肿瘤相同的组织， 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 体内诱变。具体地，细胞形成lacl/pol B -敲除小鼠 可供研究。通过评估LACL或 cll基因，我们将获得关于B- 聚合酶在正常复制和修复复制中的作用。此外，我们将 开始协调体外和体内诱变研究， 目前正在实验室进行的研究， 修复（MMR）缺陷型转基因小鼠和由其衍生的细胞系 小鼠

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.