S/MAR MCs can label cells in culture for extended periods of time and remain episomal.

<p>A) pCMV-Luc2-S/MAR PP and MC constructs were transfected into MDA-MB-231 cells, grown in the absence of antibiotic selection, and BLI was performed over the course of 9 days. On day 6, both MC and PP showed strong BLI signal within the cells. However, by day 9, the MC-labeled cells continued to display strong signal and the signal from PP-labeled cells began to disappear. On this day MC-labeled clones displaying strong luminescent signal were isolated and expanded. B) Two clones (3–5 and 3–7) were cultured over the course of 91 days post-transfection and continued to be imaged. Both clones continued to show luminescent signal over the entire 3-month period. C) Southern blot analysis was performed on total DNA isolated from control cells, from control cells spiked with 200 pg of S/MAR MC, and from an S/MAR MC clonal population (clone 3-7) 47 days after transfection. Total DNA (40 µg) was digested with a single cutting enzyme and probed with a Luc2 probe. A single band at the correct size (4.5 kb) was detectable only in the lanes with control DNA spiked with the original construct (lane 1) and the S/MAR MC clone (lane 3), confirming the episomal nature of the construct.