Development of a short-term, in vivo mutagenesis assay: the effects of methylation on the recovery of a lambda phage shuttle vector from transgenic mice.
Development of a short-term, in vivo mutagenesis assay: the effects of methylation on the recovery of a lambda phage shuttle vector from transgenic mice.
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短期体内诱变测定的开发：甲基化对从转基因小鼠中恢复 lambda 噬菌体穿梭载体的影响。
DOI：
10.1093/nar/18.10.3007
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发表时间：
1990
影响因子：
14.9
通讯作者：
Short,JM
中科院分区：
文献类型：
--
作者：
Kohler,SW;Provost,GS;Kretz,PL;Dycaico,MJ;Sorge,JA;Short,JM
通过λ噬菌体穿梭载体的稳定整合，构建了适合在染色体水平上对可疑突变原进行体内试验的转基因小鼠。穿梭载体含有/ s -半乳糖苷酶（/3-gal）靶基因，可以在体外包装提取物中从基因组DNA中获救。靶基因的突变是通过指示剂琼脂板上噬菌体斑块颜色的变化来检测的。基因组DNA的初始拯救效率低于1斑块形成单位(pfu)/100/ig，不足以进行突变分析。我们通过检测从lambda转基因动物胎儿制备的原代成纤维细胞培养物来确定低抢救效率的原因。5-氮杂环定处理的细胞的拯救效率比未处理的对照组提高了50倍，这表明甲基化抑制了拯救。甲基化的抑制作用是由观察到的。collapped菌株和mccrdeficient lambda封装提取物进一步提高了lambda拯救效率。目前超过2000 pfu/拷贝/μxg基因组DNA的救援效率比最初的救援效率提高了10万倍，允许定量突变分析。背景突变率估计为1×10−5在两个独立的谱系。用诱变剂n -乙基- rv -亚硝基脲（EtNU）处理后，小鼠脾脏DNA的突变率呈剂量依赖性增加，小鼠睾丸DNA的突变率也有显著的诱导作用
Transgenic mice suitable for thein vivoassay of uspected mutagens at the chromosome level have been constructed by stable integration of a lambda phage shuttle vector. The shuttle vector, which contains a /S-galactosidase (/3-gal) target gene, can be rescued from genomic DNA within vitropackaging extracts. Mutations In the target gene are detected by a change In lambda phage plaque color on indicator agar plates. Initial rescue efficiencies of less than 1 plaque forming unit (pfu)/100/ig of genomic DNA were too low for mutation analysis. We determined the cause of the low rescue efficiencies by examining primary fibroblast cultures prepared from fetuses of lambda transgenic animals. The rescue efficiency of 5-azacytldine-treated cells increased 50-fold over nontreated controls Indicating that methylation was inhibiting rescue. The inhibitory role of methylation was supported by the observation thatmcrdeficientE. collplating strains andmcrdeficient lambda packaging extracts further improved lambda rescue efficiency. Present rescue efficiencies of greater than 2000 pfu/copy/μxg of genomic DNA represent a 100,000-fold improvement over initial rescue efficiencies, permitting quantitative mutational analysis. The background mutagenesis rate was estimated at 1×10−5in two separate lineages. Following treatment with the mutagen N-ethyl-rV-nitrosourea (EtNU), a dose dependent increase In the mutation rate was observed in DNA isolated from mouse spleen, with significant induction also observed In mouse testes DNA
影响因子：
3.5
作者：
Justus Perthes;P. Langhans
通讯作者：
P. Langhans