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. 2013 May 28;41(14):e139. doi: 10.1093/nar/gkt442

Figure 4.

Figure 4.

Comparison of cis-acting elements for prolonged expression and minicircle maintenance. (A) Substrate BV constructs harboring different cis-acting elements. W, WPRE sequence. oriP/EBNA1 was derived from Epstein–Barr virus; S/MAR element was derived from the 5′ end of human interferon β gene; human ori was derived from 3′ end of the lamin B2 gene. (B) Duration of d2EGFP expression as measured by flow cytometry. (C) Relative minicircle copy numbers. (D) Relative BV genome copy numbers. HEK293 cells were either singly transduced with BacCdE/W (MOI 200) or BacL-CdE/W-CEO (MOI 200), or co-transduced with BacCre (MOI 100) and the substrate BV (MOI 200). The copy numbers were determined by qPCR using primers specific to the minicircle or BV genome and normalized against those at 1 dpt. (E) Examination of transgene integration by FISH. HEK293 cells were co-transduced with BacCre and BacL-CdE/W-CEO and subjected to FISH at 5 dpt using the d2egfp-specific probe. Mock-transduced cells served as negative control, whereas H-d2E cells (HEK293 cells harboring the integrated d2egfp) served as positive control. The images are representative of 100 metaphases. Scale bar, 1 µm.