Short DNA fragments induce site specific recombination in mammalian cells

Abstract

A defective hprt gene was corrected by homologous recombination in a lymphocyte cell line deficient in Hypoxanthine-phosphoribosyl-transferase activity (hprt). In a novel approach, only a fragment of a cDNA clone of the functional hprt gene was used to induce homologous recombination. The mutation that was corrected corresponds to a single base change in exon III of the hprt gene. Two transfection methods, electroporation and the previously unreported use of polyoma capsids containing only short DNA fragments, were able to induce the recombinational event. After transfection cells with a functional hprt gene were selected and homologous recombination events were identified using polymerase chain reaction. Double stranded fragments and both coding and non-coding single stranded fragments resulted in conversion to a functional gene. Analysis of the resulting hprt positive cells revealed that most cells had undergone a simple replacement reaction. Interestingly, however, some cells had lost an intron adjacent to the site of mutation. Potential mechanisms for this phenomenon, including the possible involvement of RNA in DNA repair, are discussed.