Summary
Due to their abundance and accessibility, mesothelial cells may be suitable tools for recombinant reagent expression by gene transfer. Genetically modified porcine mesothelial cells (PMCs) may have the potential for the treatment of vascular diseases in humans. We studied the effect of various transfection reagents on the primary culture of PMCs and human mesothelial cells (HMCs). The cells were transfected with a plasmid encoding a reporter gene (luciferase or green fluorescent protein [GFP]) under the control of the cytomegalovirus promoter. Transfection was achieved using cationic lipids (DOSPER and DOTAP) or calcium phosphate/deoxyribonucleic acid coprecipitation or Fugene 6. Results showed that Fugene 6 was the most efficient and reproducible transfection reagent with both PMCs and HMCs. With, Fugene 6, luciferase activity in PMCs (1.5×108 relative light units [RLU]/106 cells) was at least 2.5-fold higher than with the other transfection reagents, and it was 100-fold higher than in HMCs. However, the proportion of transfected cells expressing GFP was only 1%. These preliminary findings open up new avenues for developing experimental studies on the use of genetically modified PMCs.
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Benhamou, P. Y.; Morisoot, C.; Prevost, P.; Rolland, E.; Halimi, S.; Chroboczek, J. Standardization of procedure for efficient ex vivo gen tranfer into procine pancreatic islets with cationic liposomes. Transplantation 31:1798–1803; 1997.
Blanton Jr., J. R.; Bidwell, C. A.; Sanders, D. A.; Sharkey, C. M.; McFarland, D. C.; Gerrard, D. E.; Grant, A. L. Plasmid transfection and retroviral transduction of porcine muscle cells for cell-mediated gene transfer. J. Anim. Sci. 4:909–918; 2000.
Bordenave, L.; Remy-Zolghadri, M.; Fernandez, P.; Bareille, R.; Midy, D. Clinical performance of vascular grafts lined with endothelial cells. Endothelium 6:267–275; 1999.
Brasier, A. R.; Tate, J. E.; Habener, J. F. Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines. Biotechniques 7:1116–1123; 1989.
Brunette, E.; Stribling, R.; Debs, R. Lipofection does not require the removal of serum. Nucleic-Acids Res. 20:1151–1158; 1992.
De Wet, J. R.; Wood, K. V.; DeLuca, M.; Helinsky, D. R.; Subra Mauri, S. Firefly lucifierase gene structure and expression in mammalian cells. Mol. Cell. Biol. 7:725–737; 1987.
Dodds, W. J. The pig model for biomedical research. FASEB J. 41:247–256; 1992.
Gerrard, A. J.; Hudson, D. L.; Browlnee, G. G.; Watt, F. M. Toward gene therapy for haemophilia B using primary human keratinocytes. Nat. Genet. 3:180–183; 1993.
Ghazizadeh, S.; Harrington, R.; Garfield, J.; Taichman, L. B. Retrovirus-mediated transduction of porcine keratinocytes in organ culture. J. Invest. Dermatol. 111:492–496; 1998.
Graham, F. L.; Van Der Eb, A. J. A new technique for the assay of infectivity of human adenovirus DNA. Virology 52:456–467; 1973.
Hoff, C. M.; Cusick, J. L.; Masse, E. M.; Jackman, R. W.; Nagy, J. A.; Shcokley, T. R. Modulation of transgene expression in mesothelial cells by activation of an inducible promoter. Nephrol. Dial. Transplantation 13(6):1420–1429; 1998.
Hoff, C. M.; Shockley, T. R. The potential of gene therapy in the peritoneal cavity. Perit. Dial. Int. 19:S202-S207; 1999.
Ivarsson, M. L.; Holmdahl, L.; Falk, P.; Molne, J.; Risberg, B Characterization and fibrinolytic properties of mesothelial cells isolated from peritoneal lavage. Scand. J. Clin. Lab. Invest. 58:195–203; 1998.
Jackman, R. W.; Stapleton, T. D.; Masse, E. M.; Harvey, V. S.; Meyers, M. S.; Shockley, T. R.; Nagy, J. A. Enhancement of the functional repertoire of rat parietal peritoneal mesothelium in vivo: directed expression of the anticoagulant and antiinflammatory molecule thrombomodulin. Hum. Gen. Ther. 9:1069–1081; 1998.
Jankowski, R. J.; Wagner, W. R. Directions in cardiovascular tissue engineering. Clin. Plast. Surg. 26:605–616; 1999.
Kern, P. A.; Knedler, A.; Eckel, R. H. Isolation and culture of microvascular endothelium from human adipose tissue. J. Clin. Invest. 71:1822–1829; 1983.
Lesèche, G.; Ohan, J. Amélioration de la peméabilité des prothèses en Dacron implantées en position veineuse grace à l'ensemencement à haute densité de cellules endothéliales. Ann. Chir. Vasc. 9(3):325–326; 1995.
Murphy, J. E.; Rheinwald, J. G. Intraperitoneal injection of genetically modified human mesothelial cells for systemic gene therapy. Hum. Gene Ther. 8:1867–1879; 1997.
Nagy, J. A.; Shockley, T. R.; Jackman, R. W. Mesothelial cell gene therapy. J. Cell. Biochem. 18A (Suppl.):244; 1994.
Nagy, J. A.; Shockley, T. R.; Masse, E. M.; Harvey, V. S.; Hoff, C. M.; Jackman, R. W. Systemic delivery of recombinant protein by genetically modified mesothelial cells reseeded on the parietal peritoneal surface. Gene Ther. 2:402–410; 1995a.
Nagy, J. A.; Shockley, T. R.; Masse, E. M.; Harvey, V. S.; Jackman, R. W. Mesothelial cell mediated gene therapy: feasibility of an ex vivo strategy. Gene Ther. 2:393–401; 1995b.
Ohan, J.; Gilbert, M. A.; Brouland, J. P.; Rougier, J. P.; Trugnan, G.; Vassef, M.; Lesèche, G.; Drouet, L. Phenotypic and functional characterstics of porcine peritoneal mesothelial cells. In Vitro Cell. Dev. Biol. 35A:625–634; 1999.
Pasic, M.; Muller-Glauser, W.; Obermatt, B.,; Lachat, M.; Seifert, B.; Turina, M. Seeding with omental cells prevents late neointimal, hyperplasia in small-diameter Dacron grafts. Circulation 92:2605–2616; 1995.
Pronk, A.; Leguit, P.; Hoynch van Papendrecht, A. A.; Hagelen, E.; van Vroonhoven, T. J.; Verbrugh, H. A. A cobblestone cell isolated from the human omentum. The mesothelial cells: isolation, identification and growth characteristics. In Vitro. Cell. Dev. Biol. 29A:127–134; 1993.
Rando, T. A.; Black, H. M. Primary mouse myoblast purification characteritic and transplantation for cell mediated gene therapy. J. Cell Biol. 125:1275–1287; 1994.
Beston, J. T.; Gould-Forgerite, S.; Mannino, R. J. Potentiation of DNA mediated gene transfer in NIH3T3 cells by activators of protein kinase C. Biochim. Biophys. Acta 1088:270–276; 1991.
Rio, M. D.; Lardner, F.; Meana, A.; Segovia, J.; Alvarez, A.; Jorcano, J. Non viral transfer of gene to pig primary keratynocytes induction of angiogenesis by composite grafts of modified keratynocytes overexpression VEGF driven by a keratin promoter. Gene Ther. 10:1734–1741; 1999.
Sackman, J. E.; Freeman, M. B.; Peterseon, M. G.; Allebban, Z.; Niemeyer, G. P.; Lothrop Jr., C. D. Synthetic vascular grafts seeded with genetically modified endothelium in the dog: evaluation of the effect of seeding technique and retroviral vector on cell persistence in vivo. Cell Transplantation 2:219–235; 1995.
Schwartz, R. S.; Edward, S. D. W.; Bailey Kent, R.; Camrud, A. R.; Jorgensen, A. M. Differential neointimal response to coronary artery injury in pigs and dogs: implications for restenosis, models. Arterioscl. Thromb. 14:395–400; 1994.
Shiraishi, M.; Niroyasu, S.; Nagahama, H.; Taira, K.; Nosato, E.; Towori, H.; Obuchi, Y.; Muto, Y. Adenoviral mediated gene transfer to the porcine liver in vivo. Transplantation Proc. 7:2914–2916; 1998.
Sugawara, Y. Adenovirus-mediated transfer of tissue type plasminogen activator gene to human endothelial cells. Surgery 122:91–100; 1997.
Thomas, S. H.; Theodor, H.; Welling, B. S.; Rajabrata, S.; Messina, L. M.; Stanley, J. C. Effect of retroviral-mediated, tissue plasminogen activator gene transfer and expression on adherence and proliferation of canine endothelial cell seeded onto expanded polytetrafluoroethylene. J. Vasc. Surg. 22:795–803; 1995.
Tokeshita, S.; Losordo, D. W.; Kearney, M.; Rossow, S. T.; Isner, J. M. Time course of recombinant protein secretion after liposome-mediated gene transfer in a rabbit arterial organ culture model. Lab. Invest. 71:387–391; 1994.
van Hinsbergh, V. W.: Kooistra, T.; Scheffer, M. A.; van HajoBockel, J.; van Muijen, G. N. Characterization and fibrinolytic properties of human omental tissue mesothelial cells: comparison with endothelial cells. Blood 75:1490–1497; 1990.
Zoldhelyi, P.; McNatt, J., Shelat, H. S.; Yamamoto, Y.; Chen, Z. Q.; Willerson, J. T. Thromboresistance of balloon-injured procine carotid arteries after local gene trasfer of human tissue factor pathway inhibitor. Circulation 101:289–295; 2000.
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Ohan, J., Gilbert, M.A., Leseche, G. et al. Nonviral gene transfer into primary cultures of human and procine mesothelial cells. In Vitro Cell.Dev.Biol.-Animal 37, 402–407 (2001). https://doi.org/10.1290/1071-2690(2001)037<0402:NGTIPC>2.0.CO;2
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DOI: https://doi.org/10.1290/1071-2690(2001)037<0402:NGTIPC>2.0.CO;2