Abstract
We recently demonstrated that an RNA-DNA oligonucleotide corrected a point mutation in the mouse tyrosinase gene, resulting in permanent and inheritable restoration of tyrosinase enzymatic activity, melanin synthesis, and pigmentation changes in cultured melanocytes. In this study, we extended gene correction of melanocytes from tissue culture to live animals, using a chimeric oligonucleotide designed to correct a point mutation in the tyrosinase gene. Both topical application and intradermal injection of this oligonucleotide to albino BALB/c mouse skin resulted in dark pigmentation of several hairs in a localized area. The restored tyrosinase enzymatic activity was detected by dihydroxyphenylacetic acid (DOPA) staining of hair follicles in the treated skin. Tyrosinase gene correction was also confirmed by restriction fragment length polymorphism analysis and DNA sequencing from skin that was positive for DOPA staining and melanin synthesis. Localized gene correction was maintained three months after the last application of the chimeric oligonucleotides. These results demonstrated correction of the tyrosinase gene point mutation by chimeric oligonucleotides in vivo.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Yoon, K., Cole-Strauss, A. & Kmiec, E.B. Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA/DNA oligonucleotide. Proc. Natl. Acad. Sci. USA 93, 2071–2076 (1996).
Cole-Strauss, A. et al. Correction of the mutation responsible for sickle cell anemia by an chimeric RNA/DNA oligonucleotide. Science 273 , 1386–1389 (1996).
Xiang, Y., Cole-Strauss, A., Yoon, K., Gryn, J. & Kmiec, E.B. Targeted gene conversion in a mammalian CD34+-enriched cell population using a chimeric RNA/DNA oligonucleotide. J. Mol. Med. 75, 829–835 (1997).
Kren, B.T., Cole-Strauss, A., Kmiec, E.B. & Steer, C.J. Targeted nucleotide exchange in the alkaline phosphatase gene of HuH-7 cells mediated by a chimeric RNA/DNA oligonucleotide. Hepatology 25, 1462–1468 (1997).
Kren, B.T., Bandyopadhyay, P. & Steer, C.J. In vivo site-directed mutagenesis of the factor IX gene by chimeric RNA/DNA oligonucleotides. Nat. Med. 4, 285–290 (1998).
Alexeev, V. & Yoon, K. Stable and inheritable changes in genotype and phenotype of albino melanocytes induced by an RNA-DNA oligonucleotide . Nat. Biotechnol. 16, 1443– 1346 (1998).
Santana, E., Peritz, A.E., Iyer, S., Uitto, J. & Yoon, K. Different frequency of gene targeting events by the RNA-DNA oligonucleotide among epithelial cells. J. Invest. Dermatol. 111, 1172–1177 (1998).
Yoon, K. Single-base conversion of mammalian genes by an RNA-DNA oligonucleotide. Biog. Amines 15, 137–167 ( 1999).
Halaban, R. & Moellmann, G. White mutants in mice shedding light on humans. J. Invest. Dermatol. 100, 176S–185S (1993).
del Marmol, V. & Beermann, F. Tyrosinase and related proteins in mammalian pigmentation. FEBS Lett. 381, 165–168 (1996).
Shibahara, S. et al. A point mutation in the tyrosinase gene of BALB/c albino mouse causing the cysteine-serine substitution at position 85. Eur. J. Biochem. 189, 455–461 ( 1990).
Beermann, F. et al. Rescue of the albino phenotype by introduction of a functional tyrosinase gene into mice. EMBO J. 9, 2819– 2826 (1990).
Tanaka, S., Yamamoto, H., Takeuchi, S. & Takeuchi, T. Melanization in albino mice transformed by introducing cloned mouse tyrosinase gene. Development 108, 223– 227 (1990).
Huszar, D., Sharpe, A., Hashmi, S., Bouchard, B., Houghton, A. & Jaenisch, R. Generation of pigmented stripes in albino mice by retroviral marking of neural crest melanoblasts. Development 113, 653 –660 (1991).
Jackson, I.J. & Bennett, D.C. Identification of the albino mutation of mouse tyrosinase by analysis of an in vitro revertant. Proc. Natl. Acad. Sci. USA 87, 7010–7014 (1990).
Bennet, D.C., Cooper, P.J., Dexter, T.J., Delvin, L.M., Heasman, J. & Nester, B. Cloned mouse melanocyte line carrying the germ line mutations albino and brown: complementation in culture. Development 105, 379–385 ( 1989).
Tobin, J.D., Colen, S.R. & Bystryn, J-C. Isolation and long term cultivation of human hair follicle melanocytes. J. Invest. Dermatol. 104, 86–89 (1995).
Tobin, J.D. & Bystryn, J-C. Different population of melanocytes are present in human hair follical and epidermis. Pigm. Cell Res. 9, 304–310 ( 1996).
Narisawa, Y., Kohda, H. & Tanaka, T. Three dimension demonstration of melanocyte distribution of human hair follicles: special reference to the bulge area. Acta Derm. Venereol. 77, 97–101 (1997).
Slominski, A. & Paus, R. Melanogenesis is coupled to murine anagen: toward new concepts for the role of melanocytes and the regulation of melanogenesis in hair growth. J. Invest. Dermatol. 100, 816–822 (1993).
Slominski, A. et al. Melanogenesis during the anagen-catagen-telogen transformation of the murine hair cycle. J. Invest. Dermatol. 102, 862–869 (1994).
Straile, W.E. A study of a hair follicle and its melanocytes. Dev. Biol. 10, 45–70 (1964).
Dry, F.W. The coat of the mouse. J. Genet. 16, 287 –340 (1926).
Paus, R. & Cotsarelis, G. The biology of hair follicles . N. Engl. J. Med. 341, 491– 497 (1999).
Cotsarelis, G., Sun, T-T. & Lavker, R.M. Label-retaining cells reside in the bulge areas of pilosebaceous unit: implications for follicular stem cells, hair cycle and skin carcinogenesis . Cell 61, 1329–1337 (1990).
Li, L. & Hoffman, R.M. The feasibility of targeted gene therapy of the hair follicle. Nat. Med. 1, 705–706 (1995).
Hengge, U.R., Chan, E.F., Foster, R.A., Walker, P.S. & Vogel, J.C. Cytokine gene expression in epidermis with biological effects following injection of naked DNA. Nat. Genet. 10, 161–166 (1995).
Silvers, W.K. The coat colors of mice. (Springer-Verlag, New York; 1979).
Mason, H.S. Manual of histologic staining methods of the AFIP 3rd edn (ed. Luna, L.G.) 104–105 (McGraw-Hill, New York; 1968).
Favor, J., Neuhauser-Klaus, A., & Ehling, U.H. Radiation-induce forward and reverse specific locus mutations and dominant cataract mutations in treated strain BALB/c and DBA/2 mail mice. Mutat. Res. 177, 161– 169 (1987).
Tobin, J.D., Hagen, E., Botchkarev, V.A., & Paus R. Do hair bulb melanocytes undergo apoptosis during hair follicle regression (catagen)? J. Invest. Dermatol. 111, 941– 947 (1998).
Zhang, Z. et al. Failure to achieve gene conversion with chimeric circular oligonucleotides: potentially misleading PCR artifacts observed. Antisense Nucleic Acid Drug Dev. 8, 531–536 (1998).
Jones, P.H. & Watt, F.M. Separation of human epidermal stem cells from transit amplifying cells on the basis of differences in integrin function and expression. Cell 73, 713– 724 (1993).
Rochat, A., Kobayashi, K. & Barrandon, Y. Location of stem cells of human hair follicles by clonal analysis. Cell 76, 1063– 1073 (1994).
Acknowledgements
We thank Drs. J. Uitto and V.J. Hearing for discussion and critical reading of manuscript. This work was supported in part by grants from the NIH (AR44350; to K.Y.), and the National Alopecia Areata Foundation (to G.C.). V.A. is a recipient of a Dermatology Foundation Fellowship.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Alexeev, V., Igoucheva, O., Domashenko, A. et al. Localized in vivo genotypic and phenotypic correction of the albino mutation in skin by RNA-DNA oligonucleotide. Nat Biotechnol 18, 43–47 (2000). https://doi.org/10.1038/71901
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1038/71901
This article is cited by
-
Antisense Targeting of cFLIP Sensitizes Activated T Cells to Undergo Apoptosis and Desensitizes Responses to Contact Dermatitis
Journal of Investigative Dermatology (2009)
-
Single-strand DNA-mediated targeted mutagenesis of genomic DNA in early mouse embryos is stimulated by Rad51/54 and by Ku70/86 inhibition
Gene Therapy (2008)
-
Oligonucleotide-directed gene repair in wheat using a transient plasmid gene repair assay system
Plant Cell Reports (2006)
-
Functional improvement of mutant keratin cells on addition of desmin: an alternative approach to gene therapy for dominant diseases
Gene Therapy (2004)