xylE 转基因小鼠突变检测系统的建立  

Development of transgenic mouse mutation test system containing xylE target gene

作　　者：黄建[1,2,3] 印木泉[1,2,3] 陈耀富 陈建泉[1,2,3] 成国祥 徐少甫[1,2,3] 邱信芳 薛京伦[1,2,3] 

机构地区：[1]第二军医大学卫生毒理学教研室 [2]扬州大学农学院动物医学系 [3]复旦大学遗传学研究所

出　　处：《中国药理学与毒理学杂志》1999年第2期156-160,共5页Chinese Journal of Pharmacology and Toxicology

基　　金：上海市科学技术委员会基金

摘　　要：为了验证ｘｙｌＥ转基因小鼠能否用于体内基因突变检测，首先对从小鼠体内回收目的质粒的影响因素（电压，基因组ＤＮＡ浓度和Ｔ４连接酶浓度）进行了研究，结果发现用ＨｉｎｄⅢ酶切后的０．５μｇ组织ＤＮＡ在４００μＬ反应体系中以０．３ＵＴ４连接酶连接后，以１６ｋＶ，２００Ω，２５μＦ的参数用电穿孔法转化大肠杆菌ＤＨ１０Ｂ菌株是回收目的质粒效率最高的方法．在此基础上观察了致突变剂ｎ－甲基－ｎ′－硝基－ｎ－亚硝基胍（ＭＮＮＧ）对转基因小鼠（ｉｐ１０ｍｇｋｇ－１ｄ－１，连续４ｄ）外周血正染红细胞（ＮＣＥ）的微核率和肝组织中ｘｙｌＥ基因的自发和诱发突变率，并对突变的ｘｙｌＥ基因进行序列分析，结果ＭＮＮＧ使外周血ＮＣＥ的微核率从（２．２０±１．４８）‰上升为（６．８０±２．２８）‰（Ｐ＜０．０１），ｘｙｌＥ基因的突变率从小于１／２２８４３增至４／１８６４１，表明ＭＮＮＧ可诱发转基因小鼠ＮＣＥ微核率和肝组织中ｘｙｌＥ基因突变率显著升高．序列分析显示ＭＮＮＧ诱发的ｘｙｌＥ基因突变主要是单碱基缺失．上述结果表明ｘｙｌＥ转基因小鼠是检测体内基因突变的有效模型．To determine whether the transgenic mouse containing xylE target gene is suitable for the detection and quantification of induced mutation in vivo, the effect of field strength, genomic DNA concentration and T 4 ligase concentration on target plasmid rescue from the transgenic mouse genomic DNA were studied, it indicated that the maximum rescue frequency could be obtained when 0.5 μg HindⅢ digested genomic DNA was ligased with 0.3 U T 4 ligase in a total volume of 0.4 mL and the electroporation conditions were 16.7 kV with a 25 μF capacitor and 200 Ω in series with sample. MNNG induced(10 mg·kg 1 ·d 1 ip, for 4 d) micronucleus frequency in peripheral blood normochromatic erythrocytes (NCE) as well as mutant frequency, mutation spectrum and background mutant frequency of the G 1 transgenic mouse liver have also been characterized. The micronucleus frequency in NCE of MNNG treated mice was (6.80±2.28)‰, and that of untreated mice was (2.20±1.48)‰. There is a significant difference between them. It indicated that MNNG induced micronuclei in peripheral blood NCE of transgenic mouse. Target plasmids were recured from the MNNG treated and untreated mouse liver genomic DNA. Four xylE mutants were identified among 18641 target plasmids recured from MNNG treated mice and no mutant was identified in 22843 target plasmids from untreated mice. The spontaneous mutant frequency of the xylE target gene carried on pESnx is lower than 1/22843, the MNNG induced mutant frequency is 4/18641. The predominant mutations identified were base deletion. In conclusion the transgenic mouse containing xylE gene will be useful as in vivo system for mutagen assessment and analysis of mutagenesis mechanism.

关 键 词：xylE 转基因小鼠 突变检测系统 致突变剂 

分 类 号：R992[医药卫生—毒理学] Q319.31[医药卫生—药学]