Transposon-mediated delivery of the tetracycline inducible cassette does not improve the ability to generate clonal cell populations with an “off/on” phenotype.

<p>(A) <i>Sleeping Beauty</i> (SB) transposons encoding for inducible expression of GFP (TRP-GFP) or resistance to puromycin (PGK-Puro) were cotransfected with SB transposase (PGK-SB11) into HEK-293T expressing the tetracycline-repressor protein (Tet^R) to create cell lines that express GFP upon administration of doxycycline (Dox). (B) Flow cytometry histograms demonstrating expression of GFP in cell lines cultured in the absence (purple, No Dox) or presence (orange, Dox) of 4 μM doxycycline. Shown are representative examples of clones that were Uninduced, Leaky, Heterogenous, or Optimal with the percentage of clones for each group indicated. (C) GFP fluorescence intensity in the repressed (No Dox) and de-repressed (Plus Dox) states. *<i>P</i> = 0.0074 using Student’s <i>t</i>-test when compared to repressed (No Dox). (D) Fold increase in GFP fluorescence intensity when de-repressed (Plus Dox). Graphical representations of GFP fluorescence and fold increase were calculated from 19 clonal lines and reported as mean <u>+</u> s.e.m. TRP, Tet-responsive promoter; PGK, human phosphoglycerate kinase promoter; GFP, green fluorescent protein; SB11, <i>Sleeping Beauty</i> transposase; Puro, puromycin resistance gene; Tet^R, Tet-repressor protein; pA, poly adenylation signal.