乙基亚硝基脲诱发转基因小鼠体内基因突变及微核形成

目的 用致突变剂乙基亚硝基脲（ＥＮＵ）验证基于ＰＵＣ１１８质粒的转基因小鼠用于体内诱变研究的可行性。方法 用酶切、环化方法从经过和未经过ＥＮＵ诱变处理的ｘｙｌＥ、Ｃ５７ＢＬ／６Ｊ基因小鼠脾脏组织ＤＮＡ中重新分离ｐＵＣ１１８ＮＸ质粒,使其转化ＤＨ１０Ｂ宿主菌并铺氨苄青霉素抗性平板培养后洒邻苯二酚溶液,通过菌落颜色不同筛选突变体,并对突变靶基因,ｘｙ／Ｅ测序分析以确定突变类型。同时取该转基因小鼠外周血

Studies on gene mutation and micronucleus formation induced by ethylnitrosourea (ENU) in transgenic mice in vivo

OBJECTIVE: To determine the feasibility of using pUC 118NX plasmid-based transgenic mice in the study of induced mutation in vivo with known mutagen, ethylnitrosourea (ENU). METHODS: pUC 118NX plasmid was recovered from the spleen genomic DNA of ENU-treated and untreated xylE, C57BL/6J transgenic mice with enzyme digestion and circularizing. The recovered pUC 118NX plasmid was electroporated into DH10B host cells, which were incubated in LB solid medium containing proper ampicillin overnight and then sprayed with catechol solution. Mutant could be detected by difference in yellow and white color of the stains and its xylE target gene could be sequenced for further identifying its mutation type. In addition, peripheral blood and bone marrow cells were isolated from xylE, C57BL/6J transgenic mice and micronucleus frequency (MNF) induced by ENU was observed. RESULTS: The spontaneous mutant frequency for xylE gene in the spleen of xylE, C57BL/6J transgenic mice was less than 4.79 x 10(-5), significantly different from that treated with ENU (50 mg/kg), 19.83 x 10(-5). Types of gene mutation induced by ENU in the spleen of xylE, C57BL/6J transgenic mice included transversion (50%), one or two-base insertion (37.5%) and transition (12.5%). MNF in peripheral blood normochromatic erythrocyte (NCE) and in bone marrow polychromatic erythrocyte (PCE) of ENU-treated (50 mg/kg x 5) mice were 7.6 per thousand and 8.8 per thousand, respectively, both significantly different from the controls treated with solvent (P < 0.01). It indicated that chromosome aberration could be induced by ENU. CONCLUSION: The pUC118NX plasmid-based xylE, C57BL/6J transgenic mice, which could be used in detecting gene mutation and chromosome aberration in vivo simultaneously, provided a novel detection system for overall assessment of genetic toxicity of chemicals.