Efficient In Vitro Transfection of Human Keratinocytes with an Adenovirus-Enhanced Receptor-Mediated System  Marcel Huber, Daniel Hohl  Journal of Investigative Dermatology  Volume 114, Issue 4, Pages 661-666 (April 2000) DOI: 10.1046/j.1523-1747.2000.00942.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Optimization of SuperFect and PrimeFector systems with respect to the ratio of transfection reagents to DNA. Secondary keratinocytes in six well plates were transfected with a constant amount of pCIβgal and increasing amounts of transfection reagents as described in Materials and Methods. The following amounts of pCIβgal were used in these experiments: 0.5 μg per well for SuperFect transfections and 3 μg per well for PrimeFector transfections. The enzyme activities (units per mg) are from two independent experiments and are presented as mean ± SEM. Journal of Investigative Dermatology 2000 114, 661-666DOI: (10.1046/j.1523-1747.2000.00942.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 The AVET transfection system is more efficient than SuperFect and PrimeFector. Secondary keratinocytes in six well plates were transfected with the AVET system (1), SuperFect (2), PrimeFector (3), and mock transfection (4) using 6 μg pCIβgal per well and optimized conditions as described. Enzyme activity was measured 48 h later and expressed as a percentage of the activity obtained with the AVET system, which was set arbitrarily as 100%. Open bars refer to the specific activity calculated as units per 106 cells and hatched bars to the specific activity calculated as units per mg protein. The results are from two independent experiments and are presented as mean ± SEM. Journal of Investigative Dermatology 2000 114, 661-666DOI: (10.1046/j.1523-1747.2000.00942.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 The keratinocyte uptake of the AVET transfection complex is dependent on adenovirus receptors. (a) Human keratinocytes were transfected with the AVET system using 3 μg of pCIβgal in the presence of increasing amounts of holotransferrin in the medium. The results are shown as mean ± SEM from three independent experiments and are calculated relative to the β-galactosidase activity (set arbitrarily to 100%) of cells transfected with the same amount of pCIβgal but in the absence of holotransferrin. (b) Human keratinocytes in six well plates were transfected with 3 μg pCIβgal and AVET transfection complexes assembled with different quantities of adenovirus (0 μl black bar; 20 μl hatched bar; 40 μl open bar) and polylysine-transferrin. Enzyme activity is expressed on a logarithmic scale as units per mg protein and is presented as mean ± SEM of two independent experiments. (c) Human keratinocytes were transfected with 3 μg pCIβgal using AVET complexes in which polylysine-transferrin was replaced by the indicated quantities of polylysine. As control, a standard transfection complex with polylysine-transferrin was used, the β-galactosidase activity of which was set arbitrarily as 100%. The results are calculated as mean ± SEM from two independent experiments. (d) Human keratinocytes were transfected in the presence of chloroquine with 3 μg pCIβgal using AVET complexes prepared without adenovirus. Control cells were transfected with standard AVET complexes in the absence of chloroquine. The enzyme activities (units per mg protein) are calculated as mean ± SEM from two independent experiments. Journal of Investigative Dermatology 2000 114, 661-666DOI: (10.1046/j.1523-1747.2000.00942.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 AVET and SuperFect preferentially transfect cells adhering slowly to collagen IV. Cultured human keratinocytes were transfected with AVET and SuperFect using 3 μg pCIβgal and optimized conditions. Twenty-four hours later cells were separated by adhesion on collagen IV into RAC and SAC and then cultured an additional 24 h as described. The percentages of β-galactosidase positive cells in RAC (open bar) and SAC (hatched bar) are shown as mean ± SEM from two independent experiments. Journal of Investigative Dermatology 2000 114, 661-666DOI: (10.1046/j.1523-1747.2000.00942.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Time course of transgene expression. Keratinocytes from a TGK-negative lamellar ichthyosis patient were AVET transfected with 3 μg of pCITGK. The transglutaminase activity in the cytosolic (C) and membrane (M) fractions of transfected (T) and nontransfected (NT) cells were determined at the indicated time points. The results shown as mean ± SEM were obtained from three independent experiments. Similar time courses and magnitudes of enzyme activity were observed in secondary keratinocyte cultures obtained from other TGK-negative lamellar ichthyosis patients. Journal of Investigative Dermatology 2000 114, 661-666DOI: (10.1046/j.1523-1747.2000.00942.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions