Methods Report | Open Access
Nucleofection: A New Method for Cutaneous Gene Transfer?
Background. Transfection efficacy after nonviral gene transfer in primary epithelial cells is limited. The aim of this study was to compare transfection efficacy of the recently available method of nucleofection with the established transfection reagent FuGENE6. Methods. Primary human keratinocytes (HKC), primary human fibroblasts (HFB), and a human keratinocyte cell line (HaCaT) were transfected with reporter gene construct by FuGENE6 or Amaxa Nucleofector device. At corresponding time points, -galactosidase expression, cell proliferation (MTT-Test), transduction efficiency (X-gal staining), cell morphology, and cytotoxicity (CASY) were determined. Results. Transgene expression after nucleofection was significantly higher in HKC and HFB and detected earlier (3 h vs. 24 h) than in FuGENE6. After lipofection – of the cells remained proliferative without any influence on cell morphology. In contrast, nucleofection led to a decrease in keratinocyte cell size, with only – proliferative cells. Conclusion. Related to the method-dependent increase of cytotoxicity, transgene expression after nucleofection was earlier and higher than after lipofection.
- F Spirito, G Meneguzzi, O Danos, and M Mezzina, “Cutaneous gene transfer and therapy: the present and the future,” Journal of Gene Medicine, vol. 3, no. 1, pp. 21–31, 2001.
- T Cao, X-J Wang, and D R Roop, “Regulated cutaneous gene delivery: the skin as a bioreactor,” Human Gene Therapy, vol. 11, no. 16, pp. 2297–2300, 2000.
- P A Khavari, “Therapeutic gene delivery to the skin,” Molecular Medicine Today, vol. 3, no. 12, pp. 533–538, 1997.
- P A Khavari, “Genetic correction of inherited epidermal disorders,” Human Gene Therapy, vol. 11, no. 16, pp. 2277–2282, 2000.
- C Mah, B J Byrne, and T R Flotte, “Virus-based gene delivery systems,” Clinical Pharmacokinetics, vol. 41, no. 12, pp. 901–911, 2002.
- U R Hengge and W Bardenheuer, “Gene therapy and the skin,” American Journal of Medical Genetics Part C: Seminars in Medical Genetics, vol. 131 C, no. 1, pp. 93–100, 2004.
- T Niidome and L Huang, “Gene therapy progress and prospects: nonviral vectors,” Gene Therapy, vol. 9, no. 24, pp. 1647–1652, 2002.
- J A Wolff and V Budker, “The mechanism of naked DNA uptake and expression,” Advances in Genetics, vol. 54, pp. 3–20, 2005.
- M-P Rols, C Delteil, M Golzio, P Dumond, S Cros, and J Teissie, “In vivo electrically mediated protein and gene transfer in murine melanoma,” Nature Biotechnology, vol. 16, no. 2, pp. 168–171, 1998.
- L Steinstraesser, M Föhn, R D Klein et al., “Feasibility of biolistic gene therapy in burns,” Shock, vol. 15, no. 4, pp. 272–277, 2001.
- E Eriksson, F Yao, T Svensjö et al., “In vivo gene transfer to skin and wound by microseeding,” Journal of Surgical Research, vol. 78, no. 2, pp. 85–91, 1998.
- L B Jacobsen, S A Calvin, K E Colvin, and M Wright, “FuGENE 6 transfection reagent: the gentle power,” Methods, vol. 33, no. 2, pp. 104–112, 2004.
- J Normand and M A Karasek, “A method for the isolation and serial propagation of keratinocytes, endothelial cells, and fibroblasts from a single punch biopsy of human skin,” In Vitro Cellular and Developmental Biology—Animal, vol. 31, no. 6, pp. 447–455, 1995.
- D L Chester, D S Balderson, and R PG Papini, “A review of keratinocyte delivery to the wound bed,” Journal of Burn Care and Rehabilitation, vol. 25, no. 3, pp. 266–275, 2004.
- T Cao, S Y Tsai, B W O'Malley, X-J Wang, and D R Roop, “The epidermis as a bioreactor: topically regulated cutaneous delivery into the circulation,” Human Gene Therapy, vol. 13, no. 9, pp. 1075–1080, 2002.
- J Kopp, G Y Wang, P Kulmburg et al., “Accelerated wound healing by in vivo application of keratinocytes overexpressing KGF,” Molecular Therapy, vol. 10, no. 1, pp. 86–96, 2004.
- O Gresch, F B Engel, D Nesic et al., “New non-viral method for gene transfer into primary cells,” Methods, vol. 33, no. 2, pp. 151–163, 2004.
- A Hamm, N Krott, I Breibach, R Blindt, and A K Bosserhoff, “Efficient transfection method for primary cells,” Tissue Engineering, vol. 8, no. 2, pp. 235–245, 2002.
- P Lenz, S M Bacot, M R Frazier-Jessen, and G M Feldman, “Nucleoporation of dendritic cells: efficient gene transfer by electroporation into human monocyte-derived dendritic cells,” FEBS Letters, vol. 538, no. 1–3, pp. 149–154, 2003.
- W Martinet, D M Schrijvers, and M M Kockx, “Nucleofection as an efficient nonviral transfection method for human monocytic cells,” Biotechnology Letters, vol. 25, no. 13, pp. 1025–1029, 2003.
- H-I Trompeter, S Weinhold, C Thiel, P Wernet, and M Uhrberg, “Rapid and highly efficient gene transfer into natural killer cells by nucleofection,” Journal of Immunological Methods, vol. 274, no. 1-2, pp. 245–256, 2003.
- F Jacobsen, D Mittler, T Hirsch et al., “Transient cutaneous adenoviral gene therapy with human host defense peptide hCAP-18/LL-37 is effective for the treatment of burn wound infections,” Gene Therapy, vol. 12, no. 20, pp. 1494–1502, 2005.
- P A Khavari, O Rollman, and A Vahlquist, “Cutaneous gene transfer for skin and systemic diseases,” Journal of Internal Medicine, vol. 252, no. 1, pp. 1–10, 2002.
- P Batard, M Jordan, and F Wurm, “Transfer of high copy number plasmid into mammalian cells by calcium phosphate transfection,” Gene, vol. 270, no. 1-2, pp. 61–68, 2001.
- M Chen, W Li, J Fan, N Kasahara, and D Woodley, “An efficient gene transduction system for studying gene function in primary human dermal fibroblasts and epidermal keratinocytes,” Clinical and Experimental Dermatology, vol. 28, no. 2, pp. 193–199, 2003.
- F Jacobsen, T Hirsch, D Mittler et al., “Polybrene improves transfection efficacy of recombinant replication-deficient adenovirus in cutaneous cells and burned skin,” Journal of Gene Medicine, vol. 8, no. 2, pp. 138–146, 2006.
- R L Smith, A I Geller, K W Escudero, and C L Wilcox, “Long-term expression in sensory neurons in tissue culture from herpes simplex virus type 1 (HSV-1) promoters in an HSV-1-derived vector,” Journal of Virology, vol. 69, no. 8, pp. 4593–4599, 1995.
- I Hellgren, V Drvota, R Pieper et al., “Highly efficient cell-mediated gene transfer using-non-viral vectors and : in vitro and in vivo studies,” Cellular and Molecular Life Sciences, vol. 57, no. 8-9, pp. 1326–1333, 2000.
- U B Jensen, M S Petersen, T B Lund, T G Jensen, and L Bolund, “Transgene expression in human epidermal keratinocytes: cell cycle arrest of productively transfected cells,” Experimental Dermatology, vol. 9, no. 4, pp. 298–310, 2000.
- S H Compton, S Mecklenbeck, J E Mejia et al., “Stable integration of large ( kb) PAC constructs in HaCaT keratinocytes using an integrin-targeting peptide delivery system,” Gene Therapy, vol. 7, no. 18, pp. 1600–1605, 2000.
- N Gault, M C Vozenin-Brotons, A Calenda, J L Lefaix, and M T Martin, “Promoter sequences involved in transforming growth factor 1 gene induction in HaCat keratinocytes after gamma irradiation,” Radiation Research, vol. 157, no. 3, pp. 249–255, 2002.
- K Sumiyoshi, A Nakao, Y Setoguchi, R Tsuboi, K Okumura, and H Ogawa, “TGF-/Smad signaling inhibits IFN- and TNF--induced TARC (CCL17) production in HaCaT cells,” Journal of Dermatological Science, vol. 31, no. 1, pp. 53–58, 2003.
Copyright © 2006 Frank Jacobsen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.