Similar to other epithelial appendages, teeth form through reciprocal signaling interactions between the ectodermally derived dental epithelium and the mesenchyme. These signaling interactions program enamel matrix-secreting ameloblasts and dentin-forming odontoblasts. To study the signaling network involved in odontogenesis, we co-cultured primary human fetal dental epithelial cells and dental pulp cells to reconstitute the reciprocal signaling.
To be able to observe and analyze epithelial cells from research samples in co-culture, we tagged epithelial cells with enhanced green fluorescent protein (eGFP). Expression of eGFP was regulated by the cytokeratin 14 promoter. Cytokeratin 14, a member of the keratin family, is considered a reliable marker for dental epithelial lineage cells and basal cell compartments of epithelia.
Primary dental epithelial cells are empirically difficult to transfect due to their slow growth and vulnerability. We developed strategies to successfully deliver foreign genes into primary dental epithelial cells. In this study, we compare the use of lentivirus with FuGENE® HD Transfection Reagent to introduce a 10.5-kb eGFP reporter vector into primary dental epithelial cells.
Materials and Methods
Culture of primary dental epithelial cells
The tooth buds from 18- to 22-week old human aborted fetal tissue, obtained under the guidelines set by the University of California at San Francisco, were digested with 2 mg/ml Collagenase/Dispase for 1 hour at 37˚C. The tissue/cells were then washed with PBS, and this was followed by further digestion with STV (0.05 mM trypsin, 0.025% versene) for 5 minutes. Then, tissue/cells were pelleted and washed with PBS. Finally, 1 x 105 cells were plated on a 100-mm Primaria tissue culture dish. Epithelial cells were selected by culturing with supplemented keratinocyte medium with 0.05 mM calcium.
Construction of enhanced green fluorescence protein (eGFP) reporter vector driven by cytokeratin 14 promoter
Self-inactivated eGFP expressing lentivirus vector FUGW was used as a backbone to construct the eGFP reporter vector regulated by the human cytokeratin 14 promoter. The plasmid encoding human cytokeratin 14 promoter was provided by Nobuo Kondoh, Saitama National Defense Medical College, Japan. PCR was used to amplify cytokeratin 14 promoter and introduce PacI and BamHI cloning site. After digestion of the PCR product with PacI and BamHI, the cytokeratin 14 promoter was subcloned into FUGW to replace a human Ubiquitin-C promoter (designated as FUGW/cyto14, which is 10.5 kb).
Transfection of human dental epithelial cells with the eGFP reporter vector using FuGENE® HD Transfection Reagent
Once primary human dental epithelial cells reached 80% confluency, the medium was changed to keratinocyte base medium (KBM) without growth factors. To prepare the transfection complex, first 4 µg FUGW/cyto14 or FUGW were diluted with KBM to a final volume of 100 µl. Then, 8 µl FuGENE® HD Transfection Reagent was gently mixed with the DNA dilution and incubated for 15 minutes at room temperature. Next, the transfection complex was added to primary epithelial cells in a drop-wise manner to each well of 6-well plate containing 1 ml of medium. Twenty-four hours post transfection, expression of eGFP in dental epithelial cells was monitored using a Nikon Eclipse 300 fluorescent microscope.
Production of lentivirus and transduction of human dental epithelial cells with eGFP reporter vector using lentivirus
HEK 293 cells (human embryonic kidney cells) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. One day before transfection, the cells were trypsinized and 5 x 105 cells were plated into a well of a 6-well plate. The next morning, the cells were 90% confluent. Before transfection, the medium was changed to serum-free DMEM.
To prepare the transfection complex, first 4.4 µg FUGW/cyto14, 3.3 µg packaging vectors D8,9 and 2.3 µg VSVG were diluted with DMEM to a final volume of 100 µl. Then, 10 µl FuGENE® HD Transfection Reagent was gently mixed with the DNA dilution and incubated for 15 minutes at room temperature. Next, the transfection complex was added to 293 cells in a drop-wise manner.
Seventy-two hours after transfection, virus-containing cell medium was collected and filtered through a 0.45-µM filter. The medium was mixed with polybrene (final concentration 4 µg/ml) and added drop-wise to primary dental epithelial cells at 80% confluency. Twenty-four hours post virus infection, expression of eGFP in dental epithelial cells was monitored using a Nikon Eclipse 300 fluo-rescent microscope.
Assessment of the cytotoxicity of FuGENE® HD Transfection Reagent onprimary dental epithelial cells
Primary human dental epithelial cells were seeded into 96-well plates. Once cells reached 80% confluency, 0 µl (control), 0.2 µl, 0.4 µl, and 0.8 µl FuGENE® HD Transfection Reagent were added into the wells. Cells were incubated for another 24 hours. Then 10 µl cell proliferation reagent WST-1 was added into each well and cells were incubated for another 4 hours at 37°C. After 1 minute of vigorous shaking of the 96-well plate, the absorbance of the samples against a blank control was measured at 450 nm using a microplate reader.
Results and Discussion
In order to deliver an eGFP reporter gene into primary dental epithelial cells, researchers have been trying to optimize various transfection methods including electroporation, all of which have turned out to be inefficient. This inefficiency could be caused by the nature of primary dental epithelial cells and the size of the foreign genes to be delivered (FUGW/cyto14 is 10.5 kb).
Nevertheless, the use of lentivirus transduction is very effective, with efficiencies in primary dental epithelial cells of 50%. However, lentivirus transduction requires several additional steps to generate the virus. These steps include the cotransfection of FUGW/cyto14 with the 13.4 kb D8,9 and 6.4 kb VSVG packaging vectors into HEK 293 cells using FuGENE® HD Transfection Reagent. In addition, extra caution is necessary when handling the virus.
Direct transfection of primary dental epithelial cells with the FUGW/cyto14 using the FuGENE® HD Transfection Reagent also produced transfection efficiencies of 50% (Figure 1). The lentivirus and the FuGENE® HD Transfection Reagent methods produced the same level of transduction. As lentivirus transduction requires additional steps and reagents, using the FuGENE® HD Transfection Reagent is more time- and cost-effective.
Furthermore, a cytotoxicity assay demonstrated that the FuGENE® HD Transfection Reagent is a safe transfection reagent for sensitive primary epithelial cells. The addition of up to 0.4 µl FuGENE® HD Transfection Reagent per well did not affect the growth of the primary dental epithelial cells (Figure 2). A significant cytotoxic effect on these primary cells was detected when 0.8 µl FuGENE® HD Transfection Reagent were added to 100 µl medium in each well of a 96-well plate (Figure 2). One-way analysis of variance (ANOVA) resulted in a value of p<0.05. Tukey’s multiple comparison post-test showed a significant difference to the rest of the samples (n=4).
Our experiments demonstrated that FuGENE® HD Transfection Reagent is an effective and safe vehicle to deliver foreign genes into primary dental epithelial cells. The two methods – the viral approach with cotransfection of large vectors for preparation of lentivirus followed by lentiviral transduction of the cells and direct transfection of these cells even with a large vector of more than 10 kb – are equally efficient. Using FuGENE® HD Transfection Reagent is, however, more time- and cost-effective. Further experiments also proved that FuGENE® HD Transfection Reagent is a very effective vehicle to directly deliver genes into primary dental pulp cells (data not shown), which are difficult to transfect with other reagents.
FuGENE is a registered trademark of Fugent, L.C.C., USA. Other brands or product names are trademarks of their respective holders.
This article was originally published in Biochemica 3/2008, pages 14-16. ©Springer Medizin Verlag 2008