Figure 1

Strategy for the identification of single copy tagged high expressing cell clones for cassette exchange. (A) High expression chromosomal loci were tagged with either retrovirally or plasmid-mediated transduced expression cassettes. These cassettes were flanked by a set of heterospecific recombinase target sites. Tagged cells lines were analyzed for the stable integration of one single copy of the respective tagging vector and screened for high expressing integration loci. The transfer of a targeting vector carrying the same heterospecific recombinase target sites as the tagging vector in presence of the Flp-recombinase leads to a site-specific cassette exchange. The selection for successfully targeted clones was performed by complementing a silent, ATG-defective neomycin resistance gene pre-integrated upon tagging. This renders successfully targeted cells resistant to G418. For this purpose, the incoming targeting vector carried next to its gene of interest (antibody expression unit) a specific sequence (P/I) that facilitates expression of the neomycin resistance gene. (B) The vectors used for tagging are depicted. All tagging cassettes contain a promoter or internal ribosomal entry site for activation of the neomycin resistance gene and are flanked by heterospecific (Fwt-F5) FRT sites. Retroviral tagging was performed as described in [20] with a vector that transduces a bicistronic cassette of eGFP and a hygromycin phosphotransferase/thymidine kinase fusion protein. For plasmidic tagging vectors with different reporter genes (eGFP and/or antibody expression unit) as well as varying promoter elements (SV40/PGK) were employed. All tagging vectors express eGFP, either as a fusion protein with the hygromycin phosphotransferase/thymidine kinase or as a single protein, allowing fluorescence-based screening for the expression of the tagging cassette.