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目的：用诱变剂乙基亚硝基脲（ Ｅ Ｎ Ｕ） 验证基于ｐ Ｕ Ｃ１１８ Ｎ Ｘ 质粒载体的转基因小鼠用于研究体内基因突变的可行性。方法：用酶切、环化方式从经和未经 Ｅ Ｎ Ｕ 诱变处理的ｘｙ１ Ｅ， Ｃ５７ Ｂ Ｌ／６ Ｊ转基因小鼠脾脏组织 Ｄ Ｎ Ａ 中分离ｐ Ｕ Ｃ１１８ Ｎ Ｘ 质粒载体，使其转化 Ｄ Ｈ１０ Ｂ 宿主菌并铺 Ａｍｐｒ 平板培养后喷洒邻苯二酚溶液，通过菌落黄白颜色不同筛选突变体，并对突变靶基因ｘｙ１ Ｅ 测序分析以确定突变类型和位点。同时取该转基因小鼠外周血及骨髓细胞，观察 Ｅ Ｎ Ｕ 对转基因小鼠微核形成率的影响。结果：（１） 转基因小鼠脾脏组织ｘｙ１ Ｅ 基因自发突变率小于４ ．７９ ×１０ － ５ ，经 Ｅ Ｎ Ｕ（５０ｍ ｇ／ｋｇ ×５） 处理后，脾组织诱发基因突变率为１９ ．８３ ×１０ － ５ ，两者相差显著；（２） Ｅ Ｎ Ｕ 诱发小鼠脾组织基因突变的类型包括颠换（５０ ％ ） 、单／ 双碱基插入（３７ ．５ ％ ）和转换（１２ ．５ ％ ） ；（３） 经 Ｅ Ｎ Ｕ（５０ ｍｇ／ｋｇ ×５） 处理后，转基因小鼠外周血和骨髓细胞的微核率均明显增加，分别为７ ．６ ‰和８ ．８ ‰，与溶剂对照组相比有显著性差异（ Ｐ＜ ０ ．０１） ，表明 Ｅ Ｎ Ｕ 可明显诱发
OBJECTIVE: To verify the feasibility of using transgenic mice based on the pU C118 N X plasmid vector to study in vivo gene mutation using the mutagen E N nitrosourea (E N U). Methods: The pU C118 N X vector was isolated from the spleens of the xy1 E and C57 B L / 6 J transgenic mice by enzyme digestion and cyclization. Transformed D H10 B host strain and cultured Ampr plate after spraying catecholamine solution, by colony yellow and white color screening of different mutants, and mutant target gene xy1 E sequencing analysis to determine the type and site of mutation. At the same time, the peripheral blood and bone marrow cells of the transgenic mice were taken to observe the effect of E N U on the micronucleus formation rate in the transgenic mice. Results: (1) The spontaneous mutation rate of xy1 E gene in spleen of transgenic mice was less than 4. 79 × 10 - 5, the mutation rate of spleen induced gene was 19 after E N U (50m g / kg × 5) treatment. 83 × 10 - 5, respectively. (2) The types of gene mutations induced by E N U in mouse spleen tissues include transversion (50%), single / double base insertion .5%). (3) The micronucleus rate of peripheral blood and bone marrow cells of transgenic mice increased significantly after treated with E N U (50 mg / kg × 5), respectively. 6 ‰ and 8. 8 ‰, compared with the solvent control group was significantly different (P <0 .01), indicating that E N U can be significantly induced