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

Several GFP variants have been developedfor 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 infusion 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.