Expression and Integration of Exogenous DNA Sequences Transfected Into Mammalian Cells

Abstract

It has been previously shown that the frequency of cointegration into cellular DNA of two genes borne on the same plasmid molecule varies considerably from cell line to cell line (Colbère-Garapin et al. 1985 & 1986). In monkey kidney Vero cells, cointegration of the entire human hepatitis B surface (HBs) antigen (Ag) gene and the aminoglycoside 3’-phosphotransferase (APH3’) gene occurs only in 15 % of the transformed clones whereas in murine LM cells under the same conditions, both genes are cointegrated into 100 % of the clones (Colbère-Garapin et al. 1985 & 1986). In Vero cells, DNA rearrangements and deletions in the exogenous DNA could occur at any one of the 3 stages of the transformation process 1 — during the first days after transfection by degradation of free plasmid DNA molecules, 2 — during recombination with cell DNA and/or the early replication cycles following integration or 3 — later after integration if the transgenome is unstable. We therefore investigated the penetration, persistence and integration of cotransferred genes carried by double-stranded (ds) DNA molecules in monkey kidney Vero cells and 3 cell lines of human origin, HeLa, GM4312A and HepG2, in comparison with similarly transfected murine LM cells. Furthermore, we have compared the expression of transfected genes borne on single-stranded (ss) and ds DNA molecules in order to find out whether transient gene expression could be obtained from ss DNA, and whether stable coexpression of two genes could occur if distinct ss DNA molecules carrying each of the genes were co-transferred.