Oth the HCC and PaCa. Finally, digestion with SpeI-BfuCI is not blocked by any kind of methylation and serves as a positive control for plasmid digestion (lanes 3 and 6). These data indicate that the S/ MAR-harbouring plasmid pUbC-S/MAR is able to replicate in vivo after delivery of stably transfected cell lines, similar to studies in vitro in which S/MAR-endowed pDNA is able to achieve 3PO site mitotic stability and replication [26]. The correct size of the restriction digestion bands suggests mitotic stability without gross rearrangements of the replicating plasmid. Quantitative PCR was performed at the termination of the experiment (at 35 days post delivery) to compare the relative copy number of plasmid molecules in the Huh7 and MIA-PaCa2 treated groups. The results are shown in Figure 4C, where in bothS/MAR Vectors for In Vivo Tumour ModellingFigure 4. Molecular analysis of DNA isolated from tumour tissues at day 35 post delivery, from Huh7 and MIA-PaCa2 injected NOD/ SCID mice. (A) Southern blot analysis of pDNA isolated from two different regions of tumour tissue from NOD/SCID mice, 35days post-delivery of Huh7 and MIA-PaCa2 stable cell lines, performed as described in materials and methods. A representative hybridization pattern of pDNA isolated from one animal of each tumour is shown. Detection of indicator plasmid by M: 1-kbp ladder (Hyperladder I, Bioline); lane 1: pUbC-S/MAR isolated from the tumour tissue formed after Huh7 injection of NOD/SCID mice at 35 days post-injection; lane 2: pUbC-S/MAR isolated from a different region of the tumour tissue formed after Huh7 delivery into NOD/SCID mice at 35 days post-injection; lane 3 pUbC-S/MAR isolated from the tumour tissue formed after MIA-PaCa2 injection of NOD/SCID mice at 35 days post-injection; lane 4: pUbC-S/MAR isolated from a different region of the tumour tissue formed after MIA-PaCa2 delivery into NOD/SCID mice at 35 days post-injection; (+) positive control: 25 ng of linearized 
pUbC-S/MAR plasmid. (B) Replication-dependent assay of pUbC-S/MAR plasmid DNA isolated from the tumours of mice at 35 days post-administration. lanes 1?: Southern blot of total tumour DNA isolated from NOD/SCID mice at 35 days post-delivery with Huh7 stable cell line and 1081537 double digested with SpeI boI (lane 1), SpeI pnI (lane 2) or SpeI fuCI (lane 3) enzymes; lanes 7?: Southern of total tumour DNA isolated from NOD/SCID mice at 35 days post-delivery with MIA-PaCa2 stable cell line and double digested with SpeI boI (lane 4), SpeI pnI (lane 5) or SpeI fuCI (lane 6) enzymes; M: 1-kbp ladder (Hyperladder I, Bioline UK Ltd., London, UK). (C) Quantitative PCR performed on tumour DNA obtained at day 35 after injection of Huh7 and MIAPaCa2 cell lines. DNA was extracted from two different sites of each tumour at the end of the experiment and the number of pUbC-S/MAR vector genomes per diploid genome is shown, after normalisation with GAPDH gene, as described in materials and 
methods. (D) PCR analysis of DNA isolated in vitro from the Huh7 (lane 1) and MIA-PaCa2 (lane 4) cells before injection into NOD/SCID mice, and in vivo from two different regions of the tumour for each cell line (lanes 2,3 for Huh7 and lanes 5,6 for the MIA-PaCa2 cell lines). Expected PCR product size: 1091 bp. 100 bp DNA ladder (lane M), (+) positive control: pUbC-S/MAR; (-) negative control: PCR mix without DNA. (E) Plasmid rescue experiments of four E.Coli SC-66 colonies for Huh7 (lanes 1?) and three colonies for MIA-PaCa2 cell lines (lanes 5?),.Oth the HCC and PaCa. Finally, digestion with SpeI-BfuCI is not blocked by any kind of methylation and serves as a positive control for plasmid digestion (lanes 3 and 6). These data indicate that the S/ MAR-harbouring plasmid pUbC-S/MAR is able to replicate in vivo after delivery of stably transfected cell lines, similar to studies in vitro in which S/MAR-endowed pDNA is able to achieve mitotic stability and replication [26]. The correct size of the restriction digestion bands suggests mitotic stability without gross rearrangements of the replicating plasmid. Quantitative PCR was performed at the termination of the experiment (at 35 days post delivery) to compare the relative copy number of plasmid molecules in the Huh7 and MIA-PaCa2 treated groups. The results are shown in Figure 4C, where in bothS/MAR Vectors for In Vivo Tumour ModellingFigure 4. Molecular analysis of DNA isolated from tumour tissues at day 35 post delivery, from Huh7 and MIA-PaCa2 injected NOD/ SCID mice. (A) Southern blot analysis of pDNA isolated from two different regions of tumour tissue from NOD/SCID mice, 35days post-delivery of Huh7 and MIA-PaCa2 stable cell lines, performed as described in materials and methods. A representative hybridization pattern of pDNA isolated from one animal of each tumour is shown. Detection of indicator plasmid by M: 1-kbp ladder (Hyperladder I, Bioline); lane 1: pUbC-S/MAR isolated from the tumour tissue formed after Huh7 injection of NOD/SCID mice at 35 days post-injection; lane 2: pUbC-S/MAR isolated from a different region of the tumour tissue formed after Huh7 delivery into NOD/SCID mice at 35 days post-injection; lane 3 pUbC-S/MAR isolated from the tumour tissue formed after MIA-PaCa2 injection of NOD/SCID mice at 35 days post-injection; lane 4: pUbC-S/MAR isolated from a different region of the tumour tissue formed after MIA-PaCa2 delivery into NOD/SCID mice at 35 days post-injection; (+) positive control: 25 ng of linearized pUbC-S/MAR plasmid. (B) Replication-dependent assay of pUbC-S/MAR plasmid DNA isolated from the tumours of mice at 35 days post-administration. lanes 1?: Southern blot of total tumour DNA isolated from NOD/SCID mice at 35 days post-delivery with Huh7 stable cell line and 1081537 double digested with SpeI boI (lane 1), SpeI pnI (lane 2) or SpeI fuCI (lane 3) enzymes; lanes 7?: Southern of total tumour DNA isolated from NOD/SCID mice at 35 days post-delivery with MIA-PaCa2 stable cell line and double digested with SpeI boI (lane 4), SpeI pnI (lane 5) or SpeI fuCI (lane 6) enzymes; M: 1-kbp ladder (Hyperladder I, Bioline UK Ltd., London, UK). (C) Quantitative PCR performed on tumour DNA obtained at day 35 after injection of Huh7 and MIAPaCa2 cell lines. DNA was extracted from two different sites of each tumour at the end of the experiment and the number of pUbC-S/MAR vector genomes per diploid genome is shown, after normalisation with GAPDH gene, as described in materials and methods. (D) PCR analysis of DNA isolated in vitro from the Huh7 (lane 1) and MIA-PaCa2 (lane 4) cells before injection into NOD/SCID mice, and in vivo from two different regions of the tumour for each cell line (lanes 2,3 for Huh7 and lanes 5,6 for the MIA-PaCa2 cell lines). Expected PCR product size: 1091 bp. 100 bp DNA ladder (lane M), (+) positive control: pUbC-S/MAR; (-) negative control: PCR mix without DNA. (E) Plasmid rescue experiments of four E.Coli colonies for Huh7 (lanes 1?) and three colonies for MIA-PaCa2 cell lines (lanes 5?),.
