„GFP-Walking“: Artifical Construct Conversions caused by Simultaneous Co-Transfection analysed by Spatially and Intensity Resolved Planeometric Microscopy (SIRPM)

Several GFP variants have been developed for multicolor labeling in vivo. Here we report that simultaneous co- transfection of fluorescent protein chimeras can give falsepositive results caused by the conversion of spectral properties. The cDNA of the cysteine protease cathepsin B (CB) tagged with the enhanced yellow fluorescent protein (eYFP) and the cDNA of the histone H2A was tagged with the enhanced cyan fluorescent protein (eCFP) and were cotransfected into lung carcinoma cells. Stable clones with converted fluorescence properties were established by G418 selection and prooven on the DNA sequence level by genomic PCR. Thus, conversion is based on homologous recombination/repair/replication processes that occur between the nucleotide sequences of the fluorescent proteins. To quantify the abundance of conversion high-throughput spatially and intensity resolved planeometric microscopy (SIRPM) was applied: The fluorescent nuclei imaged with an epifluorescence microscope were segmented according to their spatial and intensity properties in both the eCFP and eYFP channels and the conversion rate was calculated from the respective number of fluorescent nuclei. 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. The conversion is independent of transfection methods or cell types. Generally, this compromises the interpretation of results obtained by dual- colour cotransfection using auto-fluorescent proteins. Consecutive transfection or low sequence similarities, however, 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.