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Nucleic Acids Research, Vol 25, Issue 23 4803-4807, Copyright © 1997 by Oxford University Press

ARTICLES

A gene trap approach to isolate mammalian genes involved in the cellular response to genotoxic stress

P Menichini, S Viaggi, E Gallerani, G Fronza, L Ottaggio, A Comes, JW Ellwart and A Abbondandolo
CSTA-Laboratory of Mutagenesis, National Institute for Research on Cancer (IST), Largo Rosanna Benzi, 10, Genoa, Italy. menikini@hp380.ist.unige.it

Treatment of cells with DNA damaging agents leads to induction of a variety of genes involved in different cellular processes. We have applied a lacZ-based gene trap strategy to search for new mammalian genes induced by genotoxic stress. A population of 32 x 10(3) neo r clones stably transfected with a gene trap vector was obtained, stained with fluorescein di-beta-d-galactopyranoside and analyzed by flow activated cell sorting and replica plating. This strategy allowed isolation of 30 neo r 'putative inducible' cell lines expressing lacZ only after a DNA damaging treatment. For three clones the site of integration and the degree of inducibility after UV treatment were determined by Southern blot and beta-galactosidase measurement respectively. One cell line (clone VI) showed a single integration site and a reproducible 3-fold induction of beta-galactosidase activity following UV irradiation. Fused transcripts were isolated from induced cells and a portion of the trapped gene was amplified by rapid amplification of cDNA ends. Sequence analysis and comparison with available gene and protein databanks revealed that the gene was novel.
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