Figure 1. Generation of BSP knockdown and control cell clones by recombinase-mediated cassette exchange.

A. The parental cell clone (a) was transfected with a plasmid containing the gene cassette for hygromycin resistance and thymidine kinase (Hyg-TK), then a hygromycin resistant cell line (b) was selected for further use. Upon gancyclovir treatment, the Hyg-TK cassette of (b) was exchanged by a cassette, in which the tet-dependent bidirectional promotor P_tet-bi controls the simultaneous expression of reporter genes firefly luciferase and red fluorescence protein mCherry as well as the miRNA targeting BSP (c). A control cell clone without miRNA (d, termed Ic1) was generated using the same strategy. Abbreviations used: hPGK – human phosphoglycerate kinase promoter; tTA – tetracycline-controlled transactivator; IRES – internal ribosome entry site; eGFP – enhanced green fluorescent protein; luciferase – firefly luciferase; P_tet-bi – bidirectional tet-regulated promoter; strawberry – red fluorescent protein strawberry; HygTK – hygromycin/ thymidine kinase selection marker gene conferring resistance to hygromycin and sensitivity to ganciclovir; pCMV – cytomegalovirus promoter; FRT and F3 - wild type and mutant Flp – recombinase target sites, B. and C: Regulation of transfected genes by doxycycline. B. Flow cytometry analysis of two double transgenic cell clones; A distinct increase in mCherry expression is observed in response to cultivation of cells in media without doxycycline; Q1-Q4 indicate quadrants 1-4; eGFP - enhanced green fluorescent protein; mCherry - red fluorescent protein mCherry; C. Western blot analysis of BSP expression in control (parent and Ic 1 clones) and two specific cell clones following six days of cultivation in media with or without doxycycline; distinct inhibition of BSP production is observed in response to cultivation of cells in media without doxycycline; numbers correspond to the following experimental conditions: 1- parent cell clone in medium with doxycycline (+dox control), 2- Ic1 cell clone in medium with dox (+dox control), 3- B3 cell clone in medium with dox (+dox specific control), 4– B7 cell clone in medium with dox (+dox specific control), 5 - parent cell clone in medium without dox (-dox control), 6- Ic1 cell clone in medium without dox (-dox control), 7- B3 (miBSP) cell clone in medium without dox (-dox anti-BSP effects), 8- B7 (miBSP) cell clone in medium without dox (-dox anti-BSP effects); D. Densitometric analysis of the respective bands from Figure 1C, based on the loading control (β – actin) and normalization of the data.