Augustin M, Sedlmeier R, Peters T, Huffstadt U, Kochmann E, Simon D, Schöniger M, Garke-Mayerthaler S, Laufs J, Mayhaus M, Franke S, Klose M, Graupner A, Kurzmann M, Zinser C, Wolf A, Voelkel M, Kellner M, Kilian M, Seelig S, Koppius A, Teubner A, Korthaus D, Nehls M, Wattler S
Ingenium Pharmaceuticals AG, Fraunhoferstr.13, 82152 Martinsried, Germany.
Mamm Genome. 2005 Jun;16(6):405-13. doi: 10.1007/s00335-004-3028-2.
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.
具有靶向基因改变的小鼠是破译生物体基因功能最有效的工具。我们构建了一个基于ENU的平行C3HeB/FeJ精子和DNA文库，其特点是有很高概率识别目标基因的等位基因变体，以及在等位基因检索和模型复苏方面具有很高效率。我们超过17000个样本的文库包含约340000个独立等位基因（每个个体样本有20个功能突变）。基于估计约30000个小鼠基因的数量，平行精子/DNA文库应能允许为每个平均目标基因识别和回收十个或更多等位基因，这意味着对于任何给定的平均基因，发现五个等位基因变体的计算成功率为99%。无关的ENU诱导的过客突变率很低，对G3代等位基因特异性表型的分析没有实际影响，因为此类无关的过客突变会被稀释并自由分离。迄今为止，已使用体外受精技术从我们的文库中获得了代表33个不同基因的39个小鼠模型。对于感兴趣基因发生突变的杂合小鼠模型，其生成时间不到2个月，即与当前基于胚胎干细胞的技术相比快三到四倍。我们得出结论，基于ENU的靶向诱变是一种强大的工具，可用于快速、高通量地生产用于生物医学研究的小鼠基因特异性模型。
