DIRECT EVIDENCE THAT TRANSGENE INTEGRATION IS RANDOM IN MURINE CELLS,IMPLYING THAT NATURALLY-OCCURRING DOUBLE-STRAND BREAKS MAY BE DISTRIBUTED SIMILARLY WITHIN THE GENOME

We have examined the distribution of illegitimate integration of a tra nsgene within the genome of cells of a murine fibroblast cell line, LT A, using fluorescence in situ hybridization (FISH) analysis. The trans gene vector contained specific sequences for detection via FISH and a hygromycin resistance gene for selection. Cells were transfected via C aPO4, and pools of 250 to 3000 hygromycin-resistant clones were subjec ted to FISH analysis. The integration of the transgene was scored for chromosome morphology (acrocentric, metacentric or dicentric) and posi tion (relative to centromere or telomere). More than 90% of the hygrom ycin-resistant clones observed involved integration of the transgene s ingly or as multiple copies, at a single site within the genome. No bi as was observed for integration of the transgene in any particular chr omosome morphology or chromosomal position, even in the presence, with in the genome, of sequences homologous to the transgene. This study pr esents direct evidence that illegitimate integration of a transgene oc curs randomly in murine fibroblasts. Since it is postulated that initi ation of illegitimate recombination involves a double-strand break (DS B), a corollary to the above results would be that naturally occurring DSBs also occur randomly within the murine genome. (C) 1998 by Radiat ion Research Society.