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. 2000 Nov;1(5):416-21.
doi: 10.1093/embo-reports/kvd089.

Genome-wide insertional mutagenesis in human cells by the Drosophila mobile element Minos

A G Klinakis  1 , L Zagoraiou, D K Vassilatis, C Savakis
Affiliations

Genome-wide insertional mutagenesis in human cells by the Drosophila mobile element Minos

A G Klinakis et al. EMBO Rep. 2000 Nov.

Abstract

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.

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Figures

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Fig. 1. Minos-derived vectors. Minos inverted terminal repeats are shown as block arrows. Arrowheads indicate the direction of transcription of the transposase (Tra) and neo genes. pA is polyadenylation signal; ori and S-P are the SV40 origin of replication and promoter, respectively.
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Fig. 2. MiLRneo insertions in individual HeLa clones. (A) Southern blot analysis of individual colonies. Genomic DNA (10 µg) was digested with the enzymes indicated before electrophoresis. The probe used is indicated by a grey bar. Arrowheads indicate the expected positions of diagnostic fragments derived from plasmid vector sequences. Lane H is from non-transfected cells. (B) Sequences flanking MiLRneo insertions. Transposon sequences and the target TA dinucleotide are in upper case.
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Fig. 3. MiLRgeo gene-trap insertions. (A) The gene-trap construct MiLRgeo. The probe used for Southern blotting is indicated by a grey bar. (B) Southern blot analysis of individual HeLa lines stably transfected with MiLRgeo. Ten micrograms of DNA were digested with EcoRV. Lane H is from non-transfected cells. (C) Fusion cDNA and flanking intron sequences of a gene-trap insertion from clone T4. Transposon sequences and the target TA dinucleotide are in upper case.
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Fig. 4. Trapping of six genes in HeLa cells with MiLRgeo. Each panel shows agarose gel electrophoresis separated, ethidium bromide stained RT–PCR products that were recovered from five independent pools (A–E) of G418-resistant clones. The control lane corresponds to a reaction without template. The panels correspond to genes (accession numbers in brackets): elongation factor 1γ (EF1γ) (Z11531); ribosomal protein 18 (rs18) (X69150); DNA replication licensing factor homolog (hRlf) (D39073); G1 to S phase transition protein 1 homolog (GSPT1) (X17644); heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP) (D28877); ATP synthase c-subunit (ATP-c) (X69908). Genes that were not detected are: thymidine kinase (M15205), ATP synthase γ-subunit (D16561), F-actin capping protein (U03271), a putative protein kinase C inhibitor (U51004), hypoxantine phosphoribosyltransferase 1 (NM_000194) and cytochrome c core I protein (L16842).
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