Fig. 1
Expression plasmid map, productivity of primary oligoclonal cell lines and long-term secretion rate dynamics of the selected clonal line. Panel a — map of the expression plasmid, CHO EEF1A1 DFR – downstream flanking area of the EEF1A1 gene, UFR – upstream flanking area; EBV TTR – fragment of the long terminal repeat from the Epstein-Barr virus; pUC ori – replication origin; bla, bla prom – ampicillin resistance gene and the corresponding promoter; CHO EEF1A1 prom, intr1 – promoter and the first intron of the EEF1A1 gene; F8BDD ORF – open reading frame of the FVIII gene with the BDD deletion; pA – polyadenylation signal. Positions of qPCR amplicons “F8B” and “ID” are marked by red and blue triangles, amplicon lengths are not to scale. Panel b – culture medium samples taken from 96-well plates, testing was done when first 10% of the wells reached confluence; panel c – selected cell lines readapted to suspension culture and tested at 1.2 × 106 cells/ml. Panel d - periodic culturing of the 11A4H cell line. Cells were passaged every third day, FVIII:C titer was measured after 3 days of culture. Stated concentrations of the MTX were used in the corresponding selection of cell cultures. Results are means, error bars represent standard deviation, n = 2. Raw data for figures are presented in corresponding Additional file 3