Integration and integrity of a bovine papillomavirus expression vector in different mammalian cells
Abstract A bovine papillomavirus expression vector carrying a neomycin resistance gene was transfected into different cultured mammalian cells (HeLa, MDCK, L6J1, NIH 3T3, C127, BHK) by the calcium phosphate precipitation method. Analysis of the state of DNA from G 418 resistant cell clones revealed that in only four (one C127 and three NIH 3T3 clones) out of 36 cell clones was the vector DNA detected as a monomeric episome. In most cases the BPV vector DNA was associated with host cell chromosomes. The vector DNA comigrated with the high molecular weight DNA fraction in agarose gels with typical integration fragments after restriction enzyme digestions. The copy numbers and integrity of the vector DNA in the cell clones were estimated by using BPV, neo Γ and pBR322 specific probes in separate quantitative hybridizations. In 25 clones clear indicators of deletions and/or rearrangements were observed. Deletions were localized in BPV and in pBR322 domains of the vector without a significant preference. The copy numbers were high in C127 clones (4–150 copies per cell), in two MDCK clones (> 100 copies per cell) and in the BHK clone (37 copies per cell). Other cell clones had five or less copies per cell. The amplified vector sequences were apparently integrated in a single chromosomal location in all except two MDCK cell clones, which had multiple integration sites.