Figure 1.
Design and construction of self-replicating minicircle constructs.
A) Vector maps of both parental plasmid (PP; top) and minicircle (MC; bottom) versions of pCMV-Luc2-S/MAR. B) Agarose gel electrophoresis analysis confirming the ability to generate both PP (8.5 kb) and MC (4.5 kb). C) Stress induced duplex destabilization (SIDD) profile of MC-pCMV-Luc2-S/MAR at a standard superhelical density of −0.05. Note the regular low denaturation energies (G(x)) between base pairs 2332 and 4314 corresponding to the location of the S/MAR motif.
Figure 2.
S/MAR MCs can label cells in culture for extended periods of time and remain episomal.
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.
Figure 3.
Luciferase activity and S/MAR MCs are slowly lost over time in labeled cell populations.
A) Normalized luciferase expression was measured at both day 64 and 121 post-transfection in three S/MAR MC clonal populations. All clones showed a trend (p = 0.18) towards decreased normalized luciferase expression over long periods of time in culture. S/MAR clone 3-7 was cultured up to day 178 after transfection and continued to show a slow loss of luciferase activity. B) The decrease in luciferase expression corresponded to a decrease in Luc2-S/MAR MC as shown via Southern blot analysis. A single band was seen in both control DNA spiked with 100 pg of S/MAR MC and DNA from S/MAR MC clone 3-7 at day 64 post-transfection. However, a band is barely discernable at day 121 from S/MAR MC clone 3-7, indicating a slow loss of S/MAR MC over time.
Figure 4.
Proliferation of S/MAR MC labeled cells can be monitored over time in living subjects.
A) S/MAR MC labeled breast cancer cells were implanted into the right flank of Nu/Nu mice and bioluminescence imaging (BLI) was performed over time. As tumors developed more luminescent signal was noted. B) This observation was confirmed by performing region of interest analysis over the tumor and measuring average radiance (p/s/cm2/sr) at days 7, 20, 28, 35 and 43 post-implantation. Significantly higher BLI signal (n = 5; * p<0.05) was noted at days 35 and 43 post-implantation compared to day 7. Error bars are S.E.M..