Genome-wide insertional mutagenesis in human cells by the Drosophila mobile element Minos
Genome-wide insertional mutagenesis in human cells by the Drosophila mobile element Minos
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DOI：
10.1093/embo-reports/kvd089
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发表时间：
2000-11-01
期刊：
影响因子：
7.7
通讯作者：
Savakis, C
中科院分区：
文献类型：
--
作者：
Klinakis, AG;Zagoraiou, L;Savakis, C
在哺乳动物细胞中开发高效的非病毒方法进行全基因组插入突变和基因标记是进行功能基因组分析的迫切需要。在这里，我们描述了转座子介导的突变(TRAMM)，使用基于果蝇Hydei转座元件Minos的裸DNA载体。通过在HeLa细胞中简单地转染质粒Minos载体，我们已经实现了高频率的细胞系的产生，每个细胞系包含一个或多个稳定的染色体整合。Minos衍生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-陷阱融合的Minos转座子在HeLa细胞中产生至少10(5)个转座子插入活性基因的HeLa细胞库，证明了在HeLa细胞中的全基因组突变。在该文库中检测到12个活性基因中的6个存在多重基因陷阱。讨论了基于Minos的TRAMM在功能基因组学中的可能应用。
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.