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A sequence-specific gene correction by an RNA-DNA oligonucleotide in mammalian cells characterized by transfection and nuclear extract using a lacZ shuttle system

Gene Therapy volume 6, pages 1960–1971 (1999)Cite this article

Abstract

The variability in gene conversion frequency by an RNA-DNA oligonucleotide (RDO) prompted us to develop a system as a means of measuring the conversion frequency rapidly and reproducibly. A shuttle vector was constructed to measure the frequency of targeted gene correction by RDO of the E. coli β-galactosidase gene containing a single point mutation (G → A), that resulted in inactivation of enzymatic activity. An RDO corrected the point mutation and restored the enzymatic activity, approximately 1%, determined by a histochemical staining in mammalian cells and by a color selection (blue or white) of bacteria transformed with Hirt DNA. In addition, we established an in vitro system capable of gene correction using nuclear extracts. CHO-K1 nuclear extracts corrected the point mutation approximately 0.1%, determined by bacterial transformation. Using the in vitro reaction, frequency of gene conversion in different cell types was measured. The embryonic fibroblasts from p53−/− mouse showed higher gene correction than that of the isogenic p53+/+ cells. Nuclear extracts from DT40 cells, which have a higher homologous recombination rate than any other mammalian cells exhibited 0.1–0.6% of gene correction. These results indicated that recombination may be rate-limiting in gene conversion by RDO in cells with competent mismatch repair activities. Utilizing transfection and in vitro reaction, we demonstrated that such a shuttle system might be useful in comparing the frequency of targeting among different cell types and to investigate the mechanism of gene conversion by RDO.

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Acknowledgements

We are grateful to Drs WE Farrell and V Alexeev for discussion and critical reading of the manuscript. This work was supported in part by the grant from the National Institute of Arthritis, Musculoskeletal and Skin Diseases (P0 AR38923 and R0 AR44350) and Dr AE Peritz was supported by a postdoctoral fellowship from the Dermatology Foundation.

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Authors and Affiliations

  1. Department of Dermatology and Cutaneous Biology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University and Jefferson Medical College, Philadelphia, 19107, PA, USA

    O Igoucheva, A E Peritz, D Levy & K Yoon

  2. Department of Biochemistry and Molecular Pharmacology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University and Jefferson Medical College, Philadelphia, 19107, PA, USA

    K Yoon

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  1. O Igoucheva
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Igoucheva, O., Peritz, A., Levy, D. et al. A sequence-specific gene correction by an RNA-DNA oligonucleotide in mammalian cells characterized by transfection and nuclear extract using a lacZ shuttle system. Gene Ther 6, 1960–1971 (1999). https://doi.org/10.1038/sj.gt.3301042

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