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Nucleic Acids Research, 1993, Vol. 21, No. 3 407-412
© 1993

MOLECULAR BIOLOGY

Targeted recombination with single-stranded DNA vectors in mammalian cells

Ken-ichiro Fujioka1,2, Yasuaki Aratani1, Kohji Kusano1,+ and Hideki Koyama1,*

1Kihara Institute for Biological Research, Yokohama City University Nakamura-cho 2-120-3, Minami-ku, Yokohama 232 2Department of Pediatrics, Yokohama City University School of Medicine 3–9 Fukuura, Kanazawa-ku, Yokohama 236, Japan

*To whom correspondence should be addressed

Received November 18, 1992. Accepted January 4, 1993.

We studied the ability of single-stranded DNA (ssDNA) to participate in targeted recombination in mammalian cells. A 5' end-deleted adenine phosphoribosyltransterase (aprt) gene was subcioned into M13 vector, and the resulting ssDNA and its double-stranded DNA (dsDNA) were transfected to APRT Chinese hamster ovary cells with a deleted aprt gene. APRT+ recombinants with the ssDNA was obtained at a frequency of 3x10–7 per survivor, which was almost equal to that with the double-stranded equivalent. Analysis of the genome in recombinant clones produced by ssDNA revealed that 12 of 14 clones resulted from correction of the deletion in the aprt locus. On the other hand, the locus of the remaining 2 was not corrected; Instead, the 5' deletion of the vector was corrected by end extension, followed by integration into random sites of the genome. To exclude the possibility that input ssDNA was converted into its duplex form before participating in a recombinatlon reaction, we compared the frequency of extrachromosomai recombination between non-complementary ssDNAs, and between one ssDNA and one dsDNA, of two phage vectors. The frequency with the ssDNAs was 0.4x10–5, being 10-fold lower than that observed with the ssDNA and the dsDNA, suggesting that as little as 10' of the transfected ssDNA was converted into duplex forms before the recombinatlon event, hence 90' remained unchanged as single-stranded molecules. Nevertheless, the above finding that ssDNA was as efficient as dsDNA in targeted recombination suggests that ssDNA itself is able to participate directly in targeted recombinatlon reactions in mammalian cells.

+Present address: Department of Molecular Biology, Institute of Medical Science, University of Tokyo, Shiroganedsi, Minato-ku, Tokyo 108, Japan


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