FIG. 3.

Effects of boundary elements on expression in the N40 locus. (A) Outline of RMCE reactions. The target vector contains an expression cassette in which the tk promoter drives expression of hygtk, a fusion protein with hygromycin phosphotransferase and thymidine kinase activities; the cassette is flanked by F and F3 elements. The five NIH clones carrying single copies of the parental construct (Fig. 2) were cotransfected with an flp-puromycin recombinase expression plasmid and a circular exchange plasmid containing the SV40 promoter driving luciferase-eGFP expression. The desired replacement was selected for by puromycin resistance, FACS, and selection against thymidine kinase expression and finally confirmed by Southern blotting. (B and C) Expression characteristics and stability of subclones derived according to the RMCE approach: expression of eGFP for locus N40 was measured for 26 passages in the absence of selection pressure and evaluated in parallel to luciferase expression (hatched bars in panel C). eGfp expression was quantified by FACS (Fig. 4), and the luciferase assay was performed on cellular extracts as described in Materials and Methods. Both reporter gene acquisition methods correlated well, confirming the expression rates in the different subclone populations. In each of the three subclones, the S/MAR-EW bordering elements caused the highest levels of eGFP and luciferase expression in comparison to other flanking DNA sequences. Between the groups of bordering elements, a significant difference in average transgene expression was observed (3 × 10⁻⁵ ≥ P ≥ 3 × 10⁻¹⁸) except between the insulator (H) and the intergenic (IS) population (P = 6 × 10⁻²). Error bars are not present in panel B, because data were obtained by a FACS-based population analysis, while panel C summarizes overall expression averages for all passages.