Genetic modification of dividing cells using episomally maintained S/MAR DNA vectors

The development of episomally maintained DNA vectors to genetically modify dividing cells efficiently and stably, without the risk of integration-mediated genotoxicity, should prove to be a valuable tool in genetic research. In this study, we demonstrate the utility of Scaffold/Matrix Attachment Region (S/MAR) DNA vectors to model the restoration of a functional wild-type copy of the gene folliculin (FLCN) implicated in the renal cancer Birt-Hogg-Dubé (BHD). Inactivation of FLCN has been shown to be involved in the development of sporadic renal neoplasia in BHD. S/MAR-modified BHD tumor cells (named UOK257-FS) show restored stable FLCN expression and have normalized downstream TGFβ signals. We demonstrate that UOK257-FS cells show a reduced growth rate in vitro and suppression of xenograft tumor development in vivo, compared with the original FLCN-null UOK257 cell line. In addition, we demonstrate that mTOR signaling in serum-starved FLCN-restored cells is differentially regulated compared with the FLCN-deficient cell. The novel UOK257-FS cell line will be useful for studying the signaling pathways affected in BHD pathogenesis. Significantly, this study demonstrates the suitability of S/MAR vectors to successfully model the functional expression of a therapeutic gene in a cancer cell line and will aid the identification of novel cancer markers for diagnosis and therapy

Genetic modification of cancer cells using non-viral, episomal S/MAR vectors for in vivo tumour modelling.

The development of genetically marked animal tumour xenografts is an area of ongoing research to enable easier and more reliable testing of cancer therapies. Genetically marked tumour models have a number of advantages over conventional tumour models, including the easy longitudinal monitoring of therapies and the reduced number of animals needed for trials. Several different methods have been used in previous studies to mark tumours genetically, however all have limitations, such as genotoxicity and other artifacts related to the usage of integrating viral vectors. Recently, we have generated an episomally maintained plasmid DNA (pDNA) expression system based on Scaffold/Matrix Attachment Region (S/MAR), which permits long-term luciferase transgene expression in the mouse liver. Here we describe a further usage of this pDNA vector with the human Ubiquitin C promoter to create stably transfected human hepatoma (Huh7) and human Pancreatic Carcinoma (MIA-PaCa2) cell lines, which were delivered into "immune deficient" mice and monitored longitudinally over time using a bioluminometer. Both cell lines revealed sustained episomal long-term luciferase expression and formation of a tumour showing the pathological characteristics of hepatocellular carcinoma (HCC) and pancreatic carcinoma (PaCa), respectively. This is the first demonstration that a pDNA vector can confer sustained episomal luciferase transgene expression in various mouse tumour models and can thus be readily utilised to follow tumour formation without interfering with the cellular genome

Molecular analysis of DNA isolated from tumour tissues at day 35 post delivery, from Huh7 and MIA-PaCa2 injected NOD/SCID mice.

<p>(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 1–3: Southern blot of total tumour DNA isolated from NOD/SCID mice at 35 days post-delivery with Huh7 stable cell line and double digested with <i>Spe</i>I–<i>Mbo</i>I (lane 1), <i>Spe</i>I–<i>Dpn</i>I (lane 2) or <i>Spe</i>I–<i>Bfu</i>CI (lane 3) enzymes; lanes 7–9: 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 <i>Spe</i>I–<i>Mbo</i>I (lane 4), <i>Spe</i>I–<i>Dpn</i>I (lane 5) or <i>Spe</i>I–<i>Bfu</i>CI (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 MIA-PaCa2 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 <i>in vitro</i> from the Huh7 (lane 1) and MIA-PaCa2 (lane 4) cells before injection into NOD/SCID mice, and <i>in vivo</i> 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 <i>E.Coli</i> colonies for Huh7 (lanes 1–4) and three colonies for MIA-PaCa2 cell lines (lanes 5–7), showing identical restriction pattern with pure pUbC-S/MAR plasmid (+), following restriction digest with <i>Spe</i>I enzyme. M: 1-kbp ladder (Hyperladder I, Bioline).</p

Histochemistry and Immunohistochemistry of tumour sections at day 35 post delivery, showing the formation of a hepatocellular carcinoma-like tumour and a pancreatic carcinoma tumour, to which luciferase expression localises.

<p>Sections from different parts of the two tumours were cut and stained with haematoxylin and eosin for histological analysis of the tumours. A–D) Sections from Huh7 injected mice. Sections have an amorphous structure and were identified as hepatocellular carcinoma (HCC) of varying degrees of differentiation: (A) Moderately differentiated HCC, magnification×10 (B–C) Sections were analysed by immunohistochemistry to show distribution of luciferase expression. Brown staining indicates luciferase positive cells. (B) Positively stained, Magnification×40 (C) Positively stained, Magnification×10 (D) Negative control: no primary antibody added, magnification×10 E–H) Sections from MIA-PaCa2 injected mice. Sections have an amorphous structure and were identified as Pancreatic carcinoma (PaCa) of varying degrees of differentiation. (E) Moderately differentiated PaCa, magnification×10 (F–G) Sections were analysed by immunohistochemistry to show distribution of luciferase expression. Brown staining indicates luciferase positive cells. (F) Positively stained, Magnification×40 (G) Positively stained, Magnification×10 (H) Negative control: no primary antibody added, magnification×10.</p

Analysis of luciferase expression from pUbC-S/MAR plasmid in stably transfected Huh7 and MIA-PaCa2 tumour cells.

<p>A) The pUbC-S/MAR plasmid used in this study, in which luciferase expression is driven by the human UbC promoter. B) Huh7, and MIA-PaCa2 cells were transfected with pUbC-S/MAR and grown under selection with G418 for about two weeks. Three single colonies were isolated and expanded out of selection with regular imaging using a Xenogen bioimager. C) Southern blot of total DNA isolated from three individual colonies for each cell line at 45 days post transfection. Lanes 1–3: Huh7 isolated colonies; Lanes 4–6 MIA-PaCa2 isolated colonies; (+): Positive control, 10 ng of bacterial pUbC-S/MAR plasmid. D) luciferase bioluminescence assay (in duplicate) on increasing amounts of Huh7 and MIA-PaCa2 cells, showing limits of signal (luciferase) detection, <i>in vitro</i>. E–F) Plasmid rescue experiments of three <i>E.Coli</i> colonies for Huh7 (lanes 1–3) and four colonies for MIA-PaCa2 cell lines (lanes 4–7), showing identical restriction pattern with pure pUbC-S/MAR plasmid (+), following restriction digest with <i>Spe</i>I enzyme. (−) negative control (no DNA); M: 1-kbp ladder (Hyperladder I, Bioline).</p