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. (B) Examples of PCR analysis. PCR was performed on total DNA extracted from liver tissues after separation of nuclei with EGFP-specific primers. Lanes 2–4 indicate three samples that tested positive (fetuses 1, 2, and 8). Hirt extracts were also analyzed by PCR. Lanes 5–7 indicate fetuses that tested positive (fetuses 9, 10, and 11). Lane 1 is a negative control (WT fetus). M, 100-bp DNA Ladder (New England Biolabs, Milan, Italy); P, positive control, pEPI-EGFP amplification.

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, Fermentas). Lane 1, control pEPI-EGFP linearized with BglII; lane 2, control pEPI-EGFP digested with BglII and EcoRI that removes the S/MAR; lane 3, rescued linearized pEPI-EGFP (BglII); lane 4, rescued double-digested plasmid (BglII and EcoRI).

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 samples. EGFP expression was compared with expression of the endogenous gene GAPDH. (B) Western blot analysis of protein extracts from a representative genetically modified fetus. EGFP protein expression from tissues of genetically modified fetus 6 and a control fetus was analyzed on 12% SDS/PAGE, blotted, and probed with anti-EGFP polyclonal antibody that recognizes a specific 26-kDa band (Upper) and with an anti-β-actin mAb that recognizes a specific 42-kDa band (Lower). One hundred fifty micrograms of protein extracts was loaded on lanes 1–6. Lane 1, WT fetus, liver; lanes 2–6, fetus 6, skeletal muscle, heart, liver, kidney, and lung. Seventy-five micrograms of protein extracts from COS7 cells transfected with pGFP-C1 was used as positive control (lane C). (C) Confocal microscopy. Micrograph of six tissues (from left to right: skeletal muscle, heart, liver, kidney, lung, and skin) from the representative positive fetus 6 (Upper) and the same tissues from the negative fetus 5 (Lower). (Magnification: ×20; bar: 20 microns.)