Klinakis A G, Zagoraiou L, Vassilatis D K, Savakis C
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Hellas, Heraklion, Greece.
EMBO Rep. 2000 Nov;1(5):416-21. doi: 10.1093/embo-reports/kvd089.
The development of efficient non-viral methodologies for genome-wide insertional mutagenesis and gene tagging in mammalian cells is highly desirable for functional genomic analysis. Here we describe transposon mediated mutagenesis (TRAMM), using naked DNA vectors based on the Drosophila hydei transposable element Minos. By simple transfections of plasmid Minos vectors in HeLa cells, we have achieved high frequency generation of cell lines, each containing one or more stable chromosomal integrations. The Minos-derived vectors insert in different locations in the mammalian genome. Genome-wide mutagenesis in HeLa cells was demonstrated by using a Minos transposon containing a lacZ-neo gene-trap fusion to generate a HeLa cell library of at least 10(5) transposon insertions in active genes. Multiple gene traps for six out of 12 active genes were detected in this library. Possible applications of Minos-based TRAMM in functional genomics are discussed.
开发高效的非病毒方法用于哺乳动物细胞全基因组插入诱变和基因标签，对于功能基因组分析而言是非常必要的。在此，我们描述了基于果蝇海德氏转座子Minos的裸DNA载体的转座子介导诱变（TRAMM）。通过在HeLa细胞中简单转染质粒Minos载体，我们实现了高频产生细胞系，每个细胞系都包含一个或多个稳定的染色体整合。源自Minos的载体插入到哺乳动物基因组的不同位置。通过使用含有lacZ-neo基因捕获融合的Minos转座子在HeLa细胞中进行全基因组诱变，以产生一个在活性基因中至少有10⁵个转座子插入的HeLa细胞文库。在该文库中检测到了12个活性基因中的6个基因的多个基因捕获。讨论了基于Minos的TRAMM在功能基因组学中的可能应用。
