Molecular Biotechnology

, Volume 33, Issue 2, pp 115–121 | Cite as

Reporter system for the detection of in vivo gene conversion

Changing colors from blue to green using GFP variants
  • Jeffrey R. Sommer
  • Jon Alderson
  • Goetz Laible
  • Robert M. Petters


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.

Index Entries

Gene conversion marker GFP BFP oligonucleotide phosphorothioated transfection 


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  1. 1.
    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.PubMedCrossRefGoogle Scholar
  2. 2.
    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.PubMedCrossRefGoogle Scholar
  3. 3.
    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.CrossRefGoogle Scholar
  4. 4.
    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.PubMedCrossRefGoogle Scholar
  5. 5.
    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.PubMedCrossRefGoogle Scholar
  6. 6.
    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.PubMedCrossRefGoogle Scholar
  7. 7.
    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.PubMedCrossRefGoogle Scholar
  8. 8.
    Igoucheva, O., Alexeev, V., and Yoon, K. (2001) Targeted gene correction by small single-stranded oligonucleotides in mammalian cells. Gene Ther. 8, 391–399.PubMedCrossRefGoogle Scholar
  9. 9.
    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.PubMedCrossRefGoogle Scholar
  10. 10.
    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.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc 2006

Authors and Affiliations

  • Jeffrey R. Sommer
    • 1
  • Jon Alderson
    • 2
  • Goetz Laible
    • 2
  • Robert M. Petters
    • 1
  1. 1.Department of Animal ScienceNorth Carolina State UniversityRaleigh
  2. 2.Agresearch Ltd, Reproductive Technologies GroupRuakura Research CenterHamiltonNew Zealand

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