Efficient and fast targeted production of murine models based on ENU mutagenesis
Efficient and fast targeted production of murine models based on ENU mutagenesis
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DOI：
10.1007/s00335-004-3028-2
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发表时间：
2005-06-01
期刊：
影响因子：
2.5
通讯作者：
Wattler, S
中科院分区：
文献类型：
--
作者：
Augustin, M;Sedlmeier, R;Wattler, S
具有靶向遗传改变的小鼠是破译生物体基因功能的最有效工具。我们生成了一个基于ENU的平行C3 HeB/FeJ精子和DNA档案，其特征在于高概率识别靶基因的等位基因变体以及等位基因检索和模型振兴的高效率。我们的档案大小超过17，000个样本，包含约340，000个独立等位基因（每个样本20个功能突变）。基于大约30，000个小鼠基因的估计数量，平行精子/DNA档案应允许每个平均靶基因鉴定和回收10个或更多个等位基因，这转化为对于任何给定的平均基因发现5个等位基因变体的计算成功率为99%。不相关的ENU诱导的乘客突变的低比率对G3代的等位基因特异性表型的分析没有实际影响，因为这种不相关的乘客突变的稀释和自由分离。迄今为止，39个小鼠模型代表33个不同的基因已恢复从我们的档案使用体外受精技术。目的基因突变杂合的鼠模型的产生时间少于2个月，即，比目前的胚胎干细胞技术快三到四倍。我们的结论是，基于ENU的靶向诱变是一个强大的工具，用于快速和高通量生产的小鼠基因特异性模型的生物医学研究。
Mice with targeted genetic alterations are the most effective tools for deciphering organismal gene function. We generated an ENU-based parallel C3HeB/FeJ sperm and DNA archive characterized by a high probability to identify allelic variants of target genes as well as high efficiencies in allele retrieval and model revitalization. Our archive size of over 17,000 samples contains approximately 340,000 independent alleles (20 functional mutations per individual sample). Based on an estimated number of approximately 30,000 mouse genes, the parallel sperm/DNA archive should permit the identification and recovery of ten or more alleles per average target gene which translates to a calculated 99% success rate in the discovery of five allelic variants for any given average gene. The low rate of unrelated ENU-induced passenger mutations has no practical impact on the analysis of the allele-specific phenotype at the G3 generation because of dilution and free segregation of such unrelated passenger mutations. To date 39 mouse models representing 33 different genes have been recovered from our archive using in vitro fertilization techniques. The generation time for a murine model heterozygous for a mutation in a gene of interest is less than 2 months, i.e., three to four times faster compared with current embryonic stem-cell-based technologies. We conclude that ENU-based targeted mutagenesis is a powerful tool for the fast and high-throughput production of murine gene-specific models for biomedical research.