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Figure 1 | BMC Biotechnology

Figure 1

From: Regulated expression of a transgene introduced on an oriP/EBNA-1 PAC shuttle vector into human cells

Figure 1

The pPAC7 vector. A) Schematic map of the pPAC7 vector which contains the EGFP gene cassette (EGFP), the blasticidin deaminase gene cassette (Bsr), the EBNA-1 gene cassette (EBNA-1), and the Epstein Barr virus origin of replication (oriP). The pUC-link fragment is removed by BamHI or NotI digestion during the cloning process and replaced by large genomic DNA fragments allowing positive selection for recombinant clones on sucrose containing media due to the SacBII gene present on the vector. The kanamycin resistance gene (Kan(R)) is a bacterial selection marker. The NotI restriction sites are used when verifying the different constructs. B) SK-BR-3 cells were transfected with pPAC7 and grown in the presence of blasticidin using the concentrations indicated. The percent of EGFP positive cells, from all viable cells, were measured by flow cytometry after 2, 4 and 6 days. The experiments were performed in triplicates with standard deviations shown. C) SK-BR-3 cells transfected with pPAC7 or pEGFP-C1 were sorted by flow cytometry and grown in non-selective medium. The percentage and total number of EGFP expressing cells was measured by flow cytometry 4, 11 and 15 days after cell sorting. The experiments were performed in triplicates with standard deviations indicated. D) The fluorescent microscopy pictures show the SK-BR-3 cells transfected with pEGFP-C1 and pPAC7 15 days after cell sorting. The shutter time is 1306 ms for pEGFP-C1 transfected and 390 ms for pPAC7 transfected cells.

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