Map of pEPI-EGFP and examples of PCR analysis. (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, is contained in the multiple cloning site (MCS). The Neo/Kan gene is driven by dual promoters to confer kanamycin resistance in bacteria and G418 resistance in mammalian cells. EGFP is the enhanced version of the GFP gene. BglII and EcoRI restriction sites are indicated. SV40, simian virus 40, ori, origin of replication, HSV, herpes simplex virus. … pEPI-EGFP is episomal in transgenic tissues. (A) Southern blot analysis. M, 1-Kbp Ladder (O’Gene Ruler, Fermentas, St. Leon-Rot, Germany), P, positive control (250 pg of linearized pEPI-EGFP vector DNA). BglII-linearized Hirt extracts from fetus 6 (liver, lane 1), fetus 9 (muscle, lane 3), and fetus 10 (kidney, lane 5, liver, lane 7) or undigested Hirt DNA from the same fetuses (lanes 2, 4, 6, and 8, respectively) are shown. (B) Example of restriction analysis of rescued plasmid (fetus 6, liver). M, 1-Kbp Ladder (O’Gene Ruler, … Analysis of EGFP expression in genetically modified fetuses. (A) RT-PCR. (Upper) M, 100-bp DNA Ladder (New England Biolabs), C, negative control. Lanes 1–5, fetuses 1, 2, 3, 6, and 8, respectively, lane 6, negative fetus 5. In every case liver was the tested organ. (Lower) M, 100-bp DNA Ladder (New England Biolabs, C, negative control, lanes 1–4 are fetuses 9, 10, 11, and 12, lane 5, negative fetus 13. Lanes C and 1, 2, and 5 are muscle samples, lanes 3 and 4 are heart … Source.