Unequal SCE is a rare event in homologous recombination between duplicated neo gene fragments in CHO cells

Abstract

The frequency of sister-chromatid exchange (SCE) was studied in Chinese hamster ovary (CHO) cell lines with stable insertions of the vector pIII-14gpt which contains 2 truncated neomycin resistance (neo) gene fragments. Recombination between regions of homology in the 2 fragments can restore a functional neo gene and make the cell resistant to the antibiotic G418, a neomycin analogue. Unequal SCE would be one of several possible mechanisms for this event. The observed spontaneous rate of formation of G418-resistant subclones was approximately 6.4 × 10 −6 per cell per generation, as compared to the estimated spontaneous frequency of 3 SCE per cell per generation. Given this SCE frequency, the probability of an SCE occurring in a target site of about 1600 bp (the distance separating the homologous regions in the neo fragments) would be about 8 × 10 −7 per cell per generation, or approximately one tenth of the estimated rate of recombination. Treatment of the cells with methyl methanesulfonate (MMS, 50 × 10 −6 M) induced about 80–90 SCE per cell, corresponding to a probability of 2 × 10 −5 SCE per 1600-bp target per cell. In the same cell culture, MMS treatment induced 4–8 × 10 −4 recombination events per cell giving rise to G418 resistance. Cells treated with HN 2 (up to 4 × 10 −6 M) showed a significant increase in SCEs, but no change in the frequency of G418-resistant revertants. These results suggest that the 2 pathways leading to SCE and recombination respectively are uncoupled, and only a small fraction of the recombination events, if any, are due to unequal SCE in this system.