AbstractThe human kidney embryonic 293 cell line (293 cells) is extensively used in biomedical and pharmaceutical research. These cells exhibit a number of numerical and structural chromosomal anomalies. However, the breakpoints responsible for these structural chromosomal rearrangements have not been comprehensively characterized. In addition, it is not known whether chromosomes with structural rearrangement are more sensitive to external toxic agents, such as ionizing radiation. We used G-banding, spectral karyotyping (SKY), and locus- and region-specific fluorescence in situ hybridization (FISH) probes designed in our lab or obtained from commercial vendor to address this gap. Our G-banding analysis revealed that the chromosome number varies from 66 to 71, with multiple rearrangements and partial additions and deletions. SKY analysis confirmed 3 consistent rearrangements, two simple and one complex in nature. Multicolor FISH analysis identified an array of breakpoints responsible for locus- and region-specific translocations. Finally, SKY analysis revealed that radio-sensitivity of structurally rearranged chromosomes is dependent on radiation dose. These findings will advance our knowledge in 293 cell biology and will enrich the understanding of radiation biology studies.