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Figure 1 | BMC Cell Biology

Figure 1

From: Establishment and mitotic stability of an extra-chromosomal mammalian replicon

Figure 1

(A) Map of pEPI-EGFP. pEPI-EGFP derives from the commercial plasmid pGFP-C1 (Clontech). An S/MAR sequence, obtained from the human IFN β-gene, was cloned into the multiple cloning site (MCS) of pGFP-C1 [7] resulting in the vector pEPI-1. The GFP gene was substituted by the enhanced version EGFP in pEPI-EGFP. The Neo/Kan gene is driven by dual promoters to confer kanamycin resistance in bacteria and G418 resistance in mammalian cells. BglII and EcoRI restriction sites are indicated. SV40, simian virus 40; ori, origin of replication; HSV, herpes simplex virus. (B) Southern blot analysis of extra-chromosomal DNA and integrated vector copies using pEPI as a probe. Lane1: 1-Kbp Ladder (O'Gene Ruler; Fermentas, St. Leon-Rot, Germany). Lane2: free extra-chromosomal pEPI DNA isolated from CHO cells transfected with supercoiled plasmid DNA, linearized by digestion with BglII demonstrating the episomal state of the vector. Lane3 and 4: DNA isolated from CHO cells transfected with linearized plasmid DNA. In this case the vector integrates at random sites into the genome and can become rearranged. DNA was digested with BglII.

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