Plasmids containing mouse rDNA do not recombine with cellular ribosomal genes when introduced into cultured mouse cells

Abstract

We have examined the fate of plasmids containing a segment of a mouse rDNA repeat after they were introduced by transfection into cultured mouse cells. In addition to the rDNA segment, the plasmids contained the thymidine kinase gene from herpes simplex virus 1 to allow for selection of the plasmid after transfection into thymidine kinase-deficient mouse cells. Thus far, no cases of homologous recombination between transfected plasmid DNAs and host cell sequences have been documented. We reasoned that the high repetition frequency of the rRNA genes in the mouse genome (200 copies per diploid cell) might create a favorable situation for obtaining homologous recombination events between the plasmids containing rDNA and host cell rDNA sequences. The plasmids were introduced into cells in both the presence and the absence of carrier DNA and both as covalently closed circles and linear molecules. The sites of plasmid integration in the genomes of various cell lines were examined by DNA restriction digests and hybridization, molecular cloning, and nuclear fractionation. In the seven cell lines examined, there was no evidence that the plasmids had integrated into the rRNA gene clusters of the cell. Thus, the apparent absence of site-specific integration of cloned DNAs introduced into mammalian cells does not appear to be due simply to the small target presented by most host cell sequences.