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. 2011 Dec 9;6(12):e27829. doi: 10.1371/journal.pone.0027829

Figure 1. Strategy and vector maps.

Figure 1

(A) Strategy for establishing production cell lines by RMCE. The tagging vector contains an EF promoter (P) controlling the expression of a GFP gene flanked by a set of heterospecific FRT sites, the synthetic variant F3 and the wild-type F. A silent, ATG-deficient neomycin resistance (Δneo) gene allows selection of targeted cells. (1) CHO Lec3.2.8.1 host cells are transfected with the tagging vector. GFP-tagged cells are isolated by two rounds of FACS. (2) Cassette exchange is initiated by co-transfecting a tagged cell line with a Flp expression vector and a targeting vector bearing the gene of interest (GOI) and a PGK promoter (P) destined to complement the Δneo gene. These genetic elements are flanked by FRT sites compatible to the tagging vector. Thus, the transiently expressed Flp recombinase exchanges the tagging gene cassette. New production cell clones with chromosomally integrated GOI are selected by G418. (B) The tagging and targeting vectors used in this study. SP = signal peptide.