Dycaico M J, Provost G S, Kretz P L, Ransom S L, Moores J C, Short J M
Stratagene, La Jolla, CA 92037.
Mutat Res. 1994 Jun 1;307(2):461-78. doi: 10.1016/0027-5107(94)90257-7.
The establishment in recent years of transgenic shuttle vector-based mutagenicity assays has provided improved systems for analysis of mutagenic and carcinogenic processes. Results in the mouse have stimulated the development of an alternate species suitable for mutation analysis and have increased our understanding of the existing models. A previously described shuttle vector (lambda LIZ), based on a lacI target gene, was constructed in this laboratory for the study of mutagenesis in transgenic mice and in cultured cell lines. The shuttle vector allows for several options in its recovery from the host genome and in mutant identification. Of the 9 transgenic lineages that were generated with the lambda LIZ vector, one was chosen for use in a standardized mutagenicity assay (Big Blue, mouse lineage A1). Characterization of this lineage included copy-number determination, chromosomal localization of transgene integration and analysis of copy-number stability. As part of the validation process, the standardized color-screening assay has been tested in the mouse, both for spontaneous mutant frequencies and with a variety of model mutagenic compounds, and has been shown to identify most major classes of mutations as evidenced by mutant spectra data. A discussion of the relative sensitivity of the shuttle vector to each of these classes of mutations is included. These studies have now been extended to the generation of transgenic rats containing the same shuttle vector for cross-species analysis. Spontaneous mutant frequencies in two transgenic rat lineages were measured in liver and in germ cells. Preliminary data suggest that spontaneous mutant frequencies in somatic tissue are lower in rats than in mice, a result consistent with historical observations of DNA damage and repair in these two species. Also under evaluation are alternative selectable systems for mutant identification, and hybrid animals obtained from mating lambda LIZ transgenics with genetically engineered mice possessing an inactivated tumor suppressor gene. It is expected that each of these widely varying endeavors will contribute, not only in furthering our understanding of the role transgenic systems should play in human risk assessment, but in illuminating the mechanisms of mutation in general.
近年来基于转基因穿梭载体的致突变性检测方法的建立，为诱变和致癌过程的分析提供了改进的系统。小鼠实验结果推动了适合突变分析的替代物种的开发，并增进了我们对现有模型的理解。本实验室构建了一种先前描述的基于lacI靶基因的穿梭载体（λLIZ），用于研究转基因小鼠和培养细胞系中的诱变作用。该穿梭载体在从宿主基因组中回收以及突变体鉴定方面有多种选择。在用λLIZ载体产生的9个转基因谱系中，选择了一个用于标准化致突变性检测（大蓝，小鼠谱系A1）。该谱系的特征包括拷贝数测定、转基因整合的染色体定位以及拷贝数稳定性分析。作为验证过程的一部分，标准化的颜色筛选检测已在小鼠中进行测试，包括自发突变频率以及使用各种模型诱变化合物进行测试，并且已证明能够识别大多数主要类型的突变，突变谱数据即为证据。文中还讨论了穿梭载体对这些类型突变中每一种的相对敏感性。这些研究现已扩展到生成含有相同穿梭载体的转基因大鼠，用于跨物种分析。在两个转基因大鼠谱系的肝脏和生殖细胞中测量了自发突变频率。初步数据表明，大鼠体细胞组织中的自发突变频率低于小鼠，这一结果与这两个物种中DNA损伤和修复的历史观察结果一致。正在评估的还有用于突变体鉴定的替代选择系统，以及通过将λLIZ转基因小鼠与具有失活肿瘤抑制基因的基因工程小鼠交配获得的杂交动物。预计这些广泛不同的研究不仅将有助于加深我们对转基因系统在人类风险评估中应发挥的作用的理解，而且将有助于阐明一般的突变机制。
