Gondo Y, Shioyama Y, Nakao K, Katsuki M
Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.
Mutat Res. 1996 May 17;360(1):1-14. doi: 10.1016/s0165-1161(96)90231-9.
To positively detect the in vivo mutations accumulated in different mouse organs, we have developed a transgenic mouse system. This transgenic mouse carried an Escherichia coli (E. coli) plasmid pML4 as a shuttle vector that consisted of a replication origin (ori), the kanamycin-resistant gene (KanR) and the rpsL+ gene (strAS) derived from E. coli. These E. coli elements were expected to be inert in the transgenic mouse system; thus, neutral mutations would be accumulated on the shuttle plasmid in the transgenic mice. The shuttle plasmid vector was recovered from the mouse genomic DNA and introduced into kanamycin-sensitive (KmS) and streptomycin-resistant (SmR) E. coli cells by using electroporation. The original pML4 shuttle plasmid transformed the host E. coli to KmR and SmS, since both the KanR and rpsL genes exhibited dominant traits of KmR and SmS, respectively. On the other hand, when the retrieved pML4 shuttle plasmid carried a mutated rpsL gene, it could be positively detected as both KmR and SmR. Based on this principle, we were able to positively detect the in vivo mutations accumulated in the rpsL transgene of the shuttle vector pML4 integrated into the mouse genome. The total number of rescued shuttle plasmids were counted on the plates containing Km alone, while only mutants were detected on the plates containing both Km and Sm. We have so far established 22 independent transgenic mouse lines that carried up to approx. 750 copies of the shuttle plasmid pML4 in a haploid genome. By using high-copy-number transgenic mouse lines which carried 350 copies or more of the shuttle vector, we also developed a simple and proficient method for retrieving the shuttle plasmid from various tissues of the transgenic mice. The background mutant frequency was approx. 5 x 10(-5). In order to validate the applicability of the positive-detection transgenic system for the induced mutagenicity assay, methylnitrosourea (MNU) was administered to the transgenic mice, and an increase in the number of mutant frequencies was seen in all tested organs including spleen, liver and brain. The rpsL transgenic mouse system was therefore considered to provide a quick-and-easy risk assessment test for in vivo tissue-specific mutagenicity, using positive detection by streptomycin.
为了阳性检测在不同小鼠器官中积累的体内突变，我们开发了一种转基因小鼠系统。这种转基因小鼠携带一个大肠杆菌（E. coli）质粒pML4作为穿梭载体，它由一个复制起点（ori）、卡那霉素抗性基因（KanR）和源自大肠杆菌的rpsL+基因（strAS）组成。这些大肠杆菌元件预计在转基因小鼠系统中是无活性的；因此，中性突变将在转基因小鼠的穿梭质粒上积累。穿梭质粒载体从小鼠基因组DNA中回收，并通过电穿孔导入对卡那霉素敏感（KmS）和对链霉素抗性（SmR）的大肠杆菌细胞中。原始的pML4穿梭质粒将宿主大肠杆菌转化为KmR和SmS，因为KanR和rpsL基因分别表现出KmR和SmS的显性性状。另一方面，当回收的pML4穿梭质粒携带一个突变的rpsL基因时，它可以被阳性检测为KmR和SmR。基于这个原理，我们能够阳性检测整合到小鼠基因组中的穿梭载体pML4的rpsL转基因中积累的体内突变。在仅含卡那霉素的平板上计数拯救的穿梭质粒总数，而在同时含卡那霉素和链霉素的平板上仅检测到突变体。到目前为止，我们已经建立了22个独立的转基因小鼠品系，其单倍体基因组中携带多达约750个穿梭质粒pML4拷贝。通过使用携带350个或更多穿梭载体拷贝的高拷贝数转基因小鼠品系，我们还开发了一种从转基因小鼠的各种组织中回收穿梭质粒的简单而有效的方法。背景突变频率约为5×10(-5)。为了验证阳性检测转基因系统在诱导诱变试验中的适用性，给转基因小鼠施用甲基亚硝基脲（MNU），并且在包括脾脏、肝脏和大脑在内的所有测试器官中都观察到突变频率的增加。因此，rpsL转基因小鼠系统被认为提供了一种使用链霉素阳性检测的体内组织特异性诱变性的快速简便风险评估测试。
