Abstract
We have devised a system for the study of in vivo gene correction based on the detection of color variants of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. The intensity and spectra of the fluorescence emitted by the blue (BFP) and red-shifted (EGFP) variants of GFP differ from each other. We modified one nucleotide from an EGFP expression vector that we predicted would yield a blue variant (TAC-CAC, Tyr66-His66). Cells that were either transiently or stably transfected with the reporter system were used to test the functionality and feasibility of the detection of in vivo gene correction. A thio-protected single-stranded oligonucleotide designed to convert the genotype of the blue variant to that of the EGFP variant by the correction of a single base pair was delivered to the reported cells using a variety of methodologies and strategies. Conversion events were easily observed using fluorescent microscopy because of the enhanced emission intensity and different spectra of the EGFP variant.
Similar content being viewed by others
References
Prasher, D. C., Eckenrode, V. K., Ward, W. W., Prendergast, F. G., and Cormier M. J. (1992) Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111, 229–233.
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., and Prasher, D. C. (1994) Green fluorescent protein as a marker for gene expression. Science 263, 802–805.
Heim, R., Prasher D. C., and Tsien R. Y. (1994) Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA 91, 12,501–12,504.
Yang, T. T., Sinai, P., Green, G. et al. (1998) Improved fluorescence and dual color detection with enhanced blue and green variants of the green fluorescent protein. J. Biol. Chem. 273, 8212–8216.
Heim, R. and Tsien, R. Y. (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr. Biol. 6, 178–182.
Igoucheva, O., Alexeev, V., and Yoon, K. (2004) Oligonucleotide-directed mutagenesis and targeted gene correction: a mechanistic point of view. Curr. Mol. Med. 4, 445–463.
Liu, L., Rice, M. C., and Kmiec, E. B. (2001) In vivo gene repair of point and frameshift mutations directed by chimetric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides. Nucleic Acids Res. 29, 4238–4250.
Igoucheva, O., Alexeev, V., and Yoon, K. (2001) Targeted gene correction by small single-stranded oligonucleotides in mammalian cells. Gene Ther. 8, 391–399.
White, P. J., Fogarty, R. D., McKean, S. C., Venables, D. J., Werther, G. A., and Wraight, C. J. (1999) Oligo-nucleotide uptake in cultured keratinocytes: influence of confluence, cationic liposomes, and keratinocyte cell type. J. Invest. Dermatol. 112, 699–705.
Radecke, F., Radecke, S., and Schwarz, K. (2004) Unmodified oligodeoxynucleotides require single-strandedness to induced targeted repair of a chromosomal EGFP gene. J. Gene Med. 6, 1257–1271.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Sommer, J.R., Alderson, J., Laible, G. et al. Reporter system for the detection of in vivo gene conversion. Mol Biotechnol 33, 115–121 (2006). https://doi.org/10.1385/MB:33:2:115
Issue Date:
DOI: https://doi.org/10.1385/MB:33:2:115