DNA-mediated alteration of the reversion frequency of transformed NIH/3T3 cells

Abstract

Cell selection immediately after DNA-mediated transfection of whole-cell DNA into mammalian cells has been used to select for specific DNA sequences that cause a phenotypic effect. Whole-cell mouse or human DNA was cleaved into a distribution of lengths (0.4-25 kilobase pairs) and transfected into anchorage-independent spontaneously transformed NIH/3T3 cells. Immediately after transaction, anchorage-dependent serum concentration-dependent reverents were selected. The Hirt supernatant, containing extrachromosomal DNA resulting from the transfection, was isolated from the revertants and transfected with high molecular weight carrier DNA into a second population of transformed cells; revertants were again selected. After five to seven cycles of transfection of Hirt supernatant DNA (obtained from revertants selected at the previous cycle) into new populations of transformed cells at each cycle, the reversion frequency had become 5-15 times greater than the spontaneous reversion frequency measured for several subclones of nontransfected or mocktransfected transformed NIH/3T3 cells. When nonmammalian genomic DNAs were used in transfecting a first population of cells, there was no effect on the reversion of frequency even after six cycles of selection. The reversion-enhancing activity of sixth-cycle Hirt supernatant DNA resulting after transfection at the first cycle with mouse or human sequences was destroyed by EcoRI but not by BamHI or Sal I. Sequences resembling human Alu I sequences were found in mouse whole-cell DNA isolated from sixth-cycle revertants generated after transfection of human sequences at the first cycle.