Different Pathways for Chromosomal Integration of Transfected Circular PSVneo Plasmids in Normal and Established Rodent Cells

The chromosomal integration of transfected circular plasmid pSV2neo molecules was investigated in normal and established mammalian fibroblasts, including two secondary cultures of normal mouse fibroblast lines, one established mouse-hamster hybrid cell line, R44, and the human fibrosarcoma line, HT1080. The physical organization of the integrated molecules was studied by restriction analysis. The results showed that whereas the normal fibroblasts predominantly integrated one head-to-tail partial dimeric molecule, the established cells predominantly integrated distinctly different molecular forms including deleted monomeres (HT1080) and various complex concatemeric molecules (R44), and frequently at more than one chromosomal site (R44). We also constructed a head-to-tail dimeric version of the plasmid, which in the case of the normal fibroblasts again integrated as a partial dimeric molecule in at least 50% of these cells. This result excluded the possibility that the normal mouse transfectants were selected for the integration of two functional neo genes. Thus, it is concluded that the distinctly different molecular forms integrated in normal and established cells demonstrate the operation of different integration pathways, the possible nature of which is discussed.