Yamada T, Yamamoto R, Kaneko H, Yoshitake A
Environmental Health Science Laboratory, Sumitomo Chemical, 1-98, 3-Chome, Kasugade-Naka, Konohana, Osaka 554-8558, Japan.
Mutat Res. 1999 Apr 26;441(1):59-72. doi: 10.1016/s1383-5718(99)00036-4.
Novel transgenic mice were developed in order to study the in vivo mutagenesis. The transgenic mice carried pCGK shuttle vector, which contained the Escherichia coli gpt gene as a mutational target, the kanamycin-resistant gene (Kanr) and cos region derived from bacteriophage lambda. The shuttle vector can be recovered from the transgenic mouse genome into the gpt-deficient E. coli by an in vitro packaging method and is selectable as a Kanr phenotype. Mutations induced at the gpt gene can be easily detected with a selective agent, 6-thioguanine (6-TG). In the previous study, the pCGK shuttle vector was incorporated into Chinese hamster CHL/IU cells and the resultant transgenic cell line was shown to be a useful system to study in vitro mutagenesis at the gpt gene. Therefore, an advantage of the shuttle vector is that in vivo mutational data obtained from the transgenic mouse can be compared with those of transgenic cell line in vitro. A transgenic CD-1 mouse line, designated as #128, that carried approximately 50 copies of pCGK shuttle vectors, was selected among 4 transgenic mouse lines. To investigate the sensitivity of the #128 line, the transgenic mice were treated with a single intraperitoneal injection of 250 mg/kg of N-ethyl-N-nitrosourea (ENU) or with 50 mg kg-1 day-1 of ENU for 5 consecutive days, and bone marrow, spleen and liver were dissected to investigate their mutational responses. The background mutant frequency was between 18x10(-6) and 75x10(-6) among all tissues tested. ENU induced significant increases in the mutant frequency above the background level in all three tissues at 14 days after single or 5-day treatment with the chemical. The increases in the mutant frequencies in bone marrow, spleen and liver were 6.4- to 6.8-fold, 3.0- to 5.6-fold and 3.0- to 3.3-fold, respectively. The shuttle vector DNA was recovered from the bone marrow of both spontaneous and ENU-treated mice and the gpt gene was amplified by polymerase chain reaction. The amplified DNA was subject to DNA sequence analysis. Out of 79 spontaneous and 52 ENU-induced mutants, the gpt gene could be amplified from 28 spontaneous and 46 ENU-induced mutants. DNA sequence analysis showed that predominant mutations were identified as A:T to T:A transversions (22 out of 46 sequenced mutants) and G:C to A:T transitions (9/46) in ENU-induced mutants, whereas G:C to T:A transversions (7 out of 28 sequenced mutants) were predominant in spontaneous mutants. These results demonstrate that this transgenic mouse, in combination with the transgenic CHL/IU cell line, is a useful system to study in vivo and in vitro mutational events at the same target gene.
为了研究体内诱变作用，构建了新型转基因小鼠。这些转基因小鼠携带pCGK穿梭载体，该载体包含作为诱变靶点的大肠杆菌gpt基因、卡那霉素抗性基因（Kanr）以及来自噬菌体λ的粘性末端（cos）区域。通过体外包装方法，穿梭载体可从转基因小鼠基因组中回收至gpt缺陷型大肠杆菌中，并可作为Kanr表型进行选择。利用选择性试剂6-硫鸟嘌呤（6-TG）可轻松检测gpt基因诱导的突变。在先前的研究中，pCGK穿梭载体被导入中国仓鼠CHL/IU细胞，所得转基因细胞系被证明是研究gpt基因体外诱变作用的有用系统。因此，穿梭载体的一个优势在于，可将从转基因小鼠获得的体内诱变数据与体外转基因细胞系的数据进行比较。在4个转基因小鼠品系中，挑选出携带约50个拷贝pCGK穿梭载体的转基因CD-1小鼠品系#128。为研究#128品系的敏感性，对转基因小鼠进行单次腹腔注射250 mg/kg的N-乙基-N-亚硝基脲（ENU），或连续5天每天注射50 mg kg-1的ENU，然后解剖骨髓、脾脏和肝脏以研究其诱变反应。在所有测试组织中，背景突变频率在18×10⁻⁶至75×10⁻⁶之间。在单次或连续5天用该化学物质处理后的第14天，ENU在所有三个组织中均诱导突变频率显著高于背景水平。骨髓、脾脏和肝脏中突变频率的增加分别为6.4至6.8倍、3.0至5.6倍和3.0至3.3倍。从自发和ENU处理小鼠的骨髓中回收穿梭载体DNA，并通过聚合酶链反应扩增gpt基因。对扩增的DNA进行DNA序列分析。在79个自发突变体和52个ENU诱导的突变体中，分别有28个自发突变体和46个ENU诱导的突变体的gpt基因可被扩增。DNA序列分析表明，在ENU诱导的突变体中，主要突变类型为A:T到T:A的颠换（46个测序突变体中有22个）和G:C到A:T的转换（9/46），而在自发突变体中，G:C到T:A的颠换（28个测序突变体中有7个）占主导。这些结果表明，这种转基因小鼠与转基因CHL/IU细胞系相结合，是研究同一靶基因体内和体外诱变事件的有用系统。
