Genome-wide insertional mutagenesis in human cells by the Drosophila mobile element Minos.
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.
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, Hellas, Heraklion, Greece.
A G
AG
Klinakis
L
L
Zagoraiou
D K
DK
Vassilatis
C
C
Savakis
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D018076:DNA, Complementary / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics*; D005091:Exons; D015723:Gene Library; D005821:Genetic Techniques; D005822:Genetic Vectors; D015894:Genome, Human; D006367:HeLa Cells; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D004251:DNA Transposable Elements; D018076:DNA, Complementary