A Gene Trap Approach to Isolate Mammalian Genes Involved in the Cellular Response to Genotoxic Stress

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 1998 Jan 24

PMID 9365260

Authors

P Menichini

S Viaggi

E Gallerani

G Fronza

L Ottaggio

A Comes

J W Ellwart

A Abbondandolo

Affiliations

Soon will be listed here.

Abstract

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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