Construct conversions caused by simultaneous co-transfection: "GFP-walking"

Felix Bestvater, Tobias A. Knoch, J÷rg Langowski, Eberhard Spiess

    Research output: Contribution to journalArticle

    9 Scopus citations

    Abstract

    Several GFP variants have been developed for multicolor labeling in vivo. Here we report that simultaneous co-transfection of fluorescent protein chimeras can give false-positive results caused by the conversion of spectral properties. Under standard transfection conditions, approximately 8% of cells produce false-positive results, but, depending on the conditions, up to 26% of the cells permanently express altered fusion proteins. This compromises the interpretation of the results. The conversion is independent of transfection methods or cell types. Our results show that the effect is based on homologous recombination/repair/replication process events that occur between the nucleotide sequences of the fluorescent proteins. Consecutive transfection or low sequence similarities avoided recombination. The appearance of conversion facilitates exchanges of spectral properties in fusion proteins, the creation of libraries, or the assembly of DNA fusion constructs in vivo. The detailed quantification of the conversion rate allows the investigation of recombination/repair/replication processes in general.

    Original languageEnglish (US)
    Pages (from-to)844-854
    Number of pages11
    JournalBioTechniques
    Volume32
    Issue number4
    StatePublished - 2002

    ASJC Scopus subject areas

    • Biotechnology
    • Biochemistry, Genetics and Molecular Biology(all)

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  • Cite this

    Bestvater, F., Knoch, T. A., Langowski, J., & Spiess, E. (2002). Construct conversions caused by simultaneous co-transfection: "GFP-walking". BioTechniques, 32(4), 844-854.