Homologous recombination between plasmids in mammalian cells can be enhanced by treatment of input DNA.
We have used the eukaryotic-prokaryotic shuttle vector pSV2Neo to demonstrate that cultured mammalian somatic cells have the enzymatic machinery to mediate homologous recombination and that the frequency of this recombination can be enhanced by pretreatment of the input DNA. Two nonoverlapping deletion mutants of pSV2Neo were constructed, each affecting the bacterial aminoglycoside 3'-phosphorylase gene (the neo gene), which confers resistance to aminoglycoside antibiotics on bacteria and resistance to the antibiotic G418 on mammalian cells. Mammalian cells transfected with either deletion plasmid alone yield no G418 -resistant colonies. Cells cotransfected with both deletion plasmids yield G418 -resistant colonies with high frequency. We show that these resistant colonies result from recombination involving homologous crossing-over or gene conversion between the deletion plasmids by rescuing from the resistant cells both types of reciprocal recombinant, full-length plasmids, and doubly deleted plasmids. Cutting one of the input plasmids to generate a double-stranded gap in the neo gene considerably enhances the frequency of homologous recombination within the gene. This suggests that targeting exogenous DNA to specific sites in mammalian chromosomes could be facilitated by suitable pretreatment of the DNA.
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D002872:Chromosome Deletion; D004262:DNA Restriction Enzymes; D004273:DNA, Neoplasm / Q000235:genetics*; D004274:DNA, Recombinant / Q000378:metabolism*; D006801:Humans; D006822:Hybrid Cells / Q000502:physiology; D007668:Kidney; D009154:Mutation; D010957:Plasmids; D014162:Transfection; D001749:Urinary Bladder Neoplasms
D004273:DNA, Neoplasm; D004274:DNA, Recombinant; D004262:DNA Restriction Enzymes