EFFICIENT RESCUE OF INTEGRATED SHUTTLE VECTORS FROM TRANSGENIC MICE - A MODEL FOR STUDYING MUTATIONS INVIVO
EFFICIENT RESCUE OF INTEGRATED SHUTTLE VECTORS FROM TRANSGENIC MICE - A MODEL FOR STUDYING MUTATIONS INVIVO
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DOI：
10.1073/pnas.86.20.7971
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发表时间：
1989-10-01
影响因子：
11.1
通讯作者：
VIJG, J
中科院分区：
文献类型：
--
作者：
GOSSEN, JA;DELEEUW, WJF;VIJG, J
为了研究实验动物不同器官和组织中的基因突变，我们培育了携带噬菌体.lambda的转基因小鼠。穿梭载体以头尾相连的方式整合到基因组中。作为诱变的靶标，选择性细菌 lacZ 基因被克隆到载体中。通过在大肠杆菌 C 的 LacZ 菌株中体外包装和繁殖噬菌体，从总基因组 DNA 中高效地拯救了整合的载体。脑和肝 DNA 中的背景突变频率似乎较低，分别从脑和肝 DNA 中分离的 138,816 和 168,160 个噬菌体中不存在无色噬菌斑表明了这一点。用N-乙基-N-亚硝基脲治疗成年雌性转基因小鼠导致从脑DNA中分离的突变载体的频率呈剂量依赖性增加，高达7.4倍。每公斤体重250毫克烷化剂为10-5。在此剂量下，在同一小鼠的肝脏DNA中，突变频率为.apprxeq。 3次。 10-5。从脑 DNA 中分离出的四种突变载体的 DNA 序列分析表明主要是 G.cntdot。 C.fwdarw。 .cntdot。 T 过渡。这些结果证明了这种转基因小鼠模型在研究体内基因突变方面的价值。除了在基础研究中的应用外，该系统还可用作敏感的、器官特异性的、短期致突变性测定。
To study gene mutations in different organs and tissues of an experimental animal, we produced transgenic mice harboring bacteriophage .lambda. shuttle vectors integrated in the genome in a head-to-tail arrangement. As a target for mutagenesis, the selectable bacterial lacZ gene was cloned in the vector. The integrated vectors were rescued from total genomic DNA with high efficiency by in vitro packaging and propagation of the phages in a LacZ- strain of Escherichia coli C. The background mutation frequencies in brain and liver DNA appeared to be low, as was indicated by the absence of colorless plaques among 138,816 and 168,160 phage isolated from brain and liver DNA, respectively. Treatment of adult female transgenic mice with N-ethyl-N-nitrosourea resulted in a dose-dependent increase of the frequency of mutated vectors isolated from brain DNA, up to 7.4 .times. 10-5 at 250 mg of the alkylating agent per kilogram of body weight. At this dose, in liver DNA of the same mice, mutation frequencies were .apprxeq. 3 .times. 10-5. DNA sequence analysis of four mutant vectors isolated from brain DNA indicated predominantly G .cntdot. C .fwdarw. A .cntdot. T transitions. These results demonstrate the value of this transgenic mouse model in studying gene mutations in vivo. In addition to its use in fundamental research, the system could be used as a sensitive, organ-specific, short-term mutagenicity assay.