We have examined the distribution of illegitimate integration of a transgene within the genome of cells of a murine fibroblast cell line, LTA, using fluorescence in situ hybridization (FISH) analysis. The transgene vector contained specific sequences for detection via FISH and a hygromycin resistance gene for selection. Cells were transfected via <tex-math>${\rm CaPO}_{4}$</tex-math>, and pools of 250 to 3000 hygromycin-resistant clones were subjected to FISH analysis. The integration of the transgene was scored for chromosome morphology (acrocentric, metacentric or dicentric) and position (relative to centromere or telomere). More than 90% of the hygromycin-resistant clones observed involved integration of the transgene singly or as multiple copies, at a single site within the genome. No bias was observed for integration of the transgene in any particular chromosome morphology or chromosomal position, even in the presence, within the genome, of sequences homologous to the transgene. This study presents direct evidence that illegitimate integration of a transgene occurs randomly in murine fibroblasts. Since it is postulated that initiation of illegitimate recombination involves a double-strand break (DSB), a corollary to the above results would be that naturally occurring DSBs also occur randomly within the murine genome.

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