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Simultaneous targeted alteration of the tyrosinase and c-kit genes by single-stranded oligonucleotides

Gene Therapy volume 9, pages 1667–1675 (2002)Cite this article

Abstract

We have shown that various forms of oligonucleotides, chimeric RNA–DNA oligonucleotide (RDO) and single-stranded oligodeoxynucleotide (ODN), are capable of chromosomal gene alterations in mammalian cells. Using two ODNs we corrected an inactivating mutation in the tyrosinase gene and introduced an activating mutation into the c-kit gene in a single albino mouse melanocyte. Relying on a pigmentation change caused by tyrosinase gene correction, we determined the frequency of gene targeting events ranging from 2×10−4 to 1×10−3, which is comparable to our previously published data using RDO. However, ODN showed more reproducible gene correction than RDO and produced pigmented cells among 60% of experiments, in comparison with 10% by RDO. DNA sequence analysis of the converted cells revealed that two out of eight individual pigmented clones harbored the mutated c-kit gene. Targeted modification of both genes resulted in the ability of the tyrosinase to convert tyrosine to melanin, and in the constitutive activation of the Kit receptor kinase. Thus, for the first time, we demonstrate the feasibility of simultaneous targeting of two genes in a single cell and show that a selection strategy to identify cells that have undergone a gene modification can enrich the targeted cells with the desired gene alteration.

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Acknowledgements

Special thanks to Dr D.C. Bennett for helpful discussions and for providing Melan-C cells. We thank Mr Steven Tutton and Ms Melissa Price for technical help. This work was supported by Dermatology Foundation Research Career Development Award for VA, and in part by grants from The National Institute of Health (GM61942, AR38923, AR44350) for KY.

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Authors and Affiliations

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

    V Alexeev, O Igoucheva & K Yoon

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

    K Yoon

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  1. V Alexeev
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  2. O Igoucheva
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  3. K Yoon
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Alexeev, V., Igoucheva, O. & Yoon, K. Simultaneous targeted alteration of the tyrosinase and c-kit genes by single-stranded oligonucleotides. Gene Ther 9, 1667–1675 (2002). https://doi.org/10.1038/sj.gt.3301862

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