Transient transfection of polarized epithelial monolayers with CFTR and reporter genes using efficacious lipids

Torry A. Tucker1,4, Karoly Varga2,4, Zsuzsa Bebok2,3,4, Akos Zsembery1,4, Nael A. McCarty5, James F. Collawn2,4, Erik M. Schwiebert1,2,4, and Lisa M. Schwiebert1,2,4

Departments of 1 Physiology and Biophysics, 2 Cell Biology, and 3 Medicine and the 4 Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, 35294-0005; and 5 School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332-0230


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Transient transfection of epithelial cells with lipid reagents has been limited because of toxicity and lack of efficacy. In this study, we show that more recently developed lipids transfect nonpolarized human airway epithelial cells with high efficacy and efficiency and little or no toxicity. Because of this success, we hypothesized that these lipids may also allow transient transfection of polarized epithelial monolayers. A panel of reagents was tested for transfer of the reporter gene luciferase (LUC) into polarized monolayers of non-cystic fibrosis (non-CF) and CF human bronchial epithelial cells, MDCK epithelial cell monolayers, and, ultimately, primary non-CF and CF airway epithelial cells. Lipid reagents, which were most successful in initial LUC assays, were also tested for transfer of vectors bearing the reporter gene green fluorescent protein (GFP) and for successful transfection and expression of an epithelial-specific protein, the cystic fibrosis transmembrane conductance regulator (CFTR). Electrophysiological, biochemical, and immunological assays were performed to show successful complementation of an epithelial monolayer with transiently expressed CFTR. We also present findings that help facilitate monolayer formation by these airway epithelial cell lines. Together, these data show that polarized monolayers are transfected transiently with more recently developed lipids, specifically LipofectAMINE PLUS and LipofectAMINE 2000. Transient transfection of epithelial monolayers provides a powerful system in which to express the cDNA of any epithelium-specific protein transiently in a native polarized epithelium to study protein function.

epithelia; lipid-mediated gene transfer; cystic fibrosis transmembrane conductance regulator; cystic fibrosis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

TRANSIENT TRANSFECTION of epithelial cells with original lipid reagents such as Lipofectin or LipofectAMINE is limited in efficacy in epithelial cells grown in nonpolarized culture. However, in this study our laboratories have shown that more recently developed lipid compounds are quite efficacious in transiently transfecting non-cystic fibrosis (non-CF) and CF airway epithelial cell lines. In particular, LipofectAMINE PLUS and LipofectAMINE 2000 are the most efficacious and showed little or no toxicity. In early stages of this work, two studies by our laboratories used this emerging technology on nonpolarized epithelial and heterologous cells (1, 5). Because of this breakthrough in epithelial "lipofection" efficiency, we hypothesized that these more newly developed lipid transfecting reagents might also prove efficacious in transducing polarized epithelial monolayers.

Few studies have shown successful transfection of polarized epithelia. Snyder (6) recently reported a study in which alpha -, beta -, and gamma -isoforms of the epithelial sodium channel (ENaC) were expressed together and transiently in Fischer rat thyroid epithelial cells grown as polarized monolayers. Transient transfection was undertaken on forming monolayers that did not disrupt transepithelial resistance and resulted in expression of the ENaC constructs for 3-7 days following transfection (6). Both electrophysiology and biochemistry could be performed on these polarized monolayers (6). The lipid reagent mixture used by Snyder was proprietary, but it contained DOPE- and DMRIE-based cationic lipids. Moreover, Li and Mrsny (3) transiently transfected a rat salivary gland epithelial cell line grown as a monolayer with LipofectAMINE PLUS and cDNAs encoding the proteins Raf-1, occludin, and claudin-1 to affect tight junction and monolayer integrity. Uduehi et al. (9) transiently transfected Caco-2 intestinal epithelial monolayers with DNA:DOTAP complexes early during monolayer formation, although the Caco-2 monolayers became resistant to lipid-mediated transfection as they aged.

Based on our previous results and the handful of studies outlined above, our working hypothesis was that newly developed lipid reagents, especially those that used an enhancer or priming lipid to complex with mammalian expression vectors before mixing with transfecting lipid (LipofectAMINE PLUS, for example), may have efficacy in transiently transfecting epithelial cell monolayers. The data described herein show that mammalian expression vectors bearing the reporter genes luciferase (LUC) or green fluorescent protein (GFP) or the epithelium-specific gene, the cystic fibrosis transmembrane conductance regulator (CFTR), are transfected transiently into forming epithelial monolayers without damage or toxicity to the monolayer and with positive gene transduction. This work derives from efficient gene transfer in nonpolarized human airway epithelial cells. These results as well as the establishment of these transfection systems may have wide utility to investigators who wish to study the activity of their epithelial protein of interest in a polarized epithelium. These systems may also obviate the need for establishing stably transfected clones, where levels of gene and protein expression vary widely from clone to clone or in which the loss of expression over passage or time in culture may occur.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell and Monolayer Culture

Culture in flasks. All substrates for all epithelial cell lines were coated with Vitrogen 100 solution (Cohesion) that was diluted 1:15 in Ca2+,Mg2+-free Dulbecco's PBS (Life Technologies, Grand Island, NY). The diluted Vitrogen was added to the substrate and incubated for 1-2 min before removal. For filter supports, the incubation was 5-10 min. Coating was performed a minimum of 8 h before plating of epithelial cells. Four epithelial cell lines were used for this study: a 16HBE14o- non-CF human bronchial epithelial cell line and a CFBE41o- CF human bronchial epithelial cell line (2, 10); an MDCK C7 canine kidney epithelial cell line (4); an IB3-1 CF human bronchial epithelial cell line (12); and a CFT-1 CF human tracheal epithelial cell line (11). MDCK cells, CFBE41o- cells, and 16HBE14o- cells were grown in tissue culture flasks in a minimum essential medium (MEM) with Earle's salts and L-glutamine (Life Technologies) supplemented with 5% fetal bovine serum (certified FBS, heat-inactivated at 55°C for 30 min; Life Technologies), 6 ml of penicillin-streptomycin 100× solution (penicillin 100 U/ml final; streptomycin 100 µg/ml final; Life Technologies), 6 ml of 200 mM L-glutamine 100× solution (2 mM final; Life Technologies), 2 ml of fungizone solution (amphotericin B, 250 µg/ml stock, 1 µg/ml final; Life Technologies), and 1 ml of gentamycin stock solution (100 µg/ml final; Life Technologies). IB3-1 cells were grown in LHC-8 medium (Biofluids, Rockville, MD) supplemented with 5% FBS, 10 ml of penicillin-streptomycin 100× solution, 5 ml of 200 mM L-glutamine 100× solution (2 mM final; Life Technologies), and 1 ml of fungizone solution. CFT-1 cells were grown in the same LHC-8 medium used for IB3-1 cells, the only exception being no FBS supplementation. CFT-1 and CFBE41o- cells are homozygous for the Delta F508-CFTR mutation, whereas IB3-1 bears the Delta F508-CFTR and W1282X-CFTR mutations.

Culture on permeable filter supports. For 16HBE14o-, CFBE41o-, IB3-1, and CFT-1 airway epithelial cells, cells were seeded onto 6.5-mm-diameter Transwell filters (Corning-Costar, Corning, NY). Despite standardized seeding of cells (1 × 106 cells per 6.5-mm-diameter filter; 2× cells for 12-mm-diameter filters; 4× cells for 24-mm-diameter filters), transepithelial resistance (RTE) was always greatest for the airway epithelial cell lines on the 6.5-mm-diameter filters. RTE was routinely twofold greater on clear polyester Transwell filter supports than on more standard polycarbonate Transwell filters. For MDCK cells, the same findings also held true; however, sufficient RTE values were observed on the different size filters (3,000-6,000 Omega  · cm2 for 6.5-mm filter monolayers; 1,000-2,000 Omega  · cm2 for 12-mm filter monolayers; 500-1,000 Omega  · cm2 for 24-mm filter monolayers). Data from different size filters are shown for MDCK cells and is indicated in the figure legends. Lots of certified FBS are routinely screened by our laboratory for optimal support of 16HBE14o- cell monolayer formation and are routinely available from Life Technologies (now a division of Invitrogen). These lots of FBS are key ingredients in helping the epithelial cell lines establish polarized monolayers. Other lots of FBS from Life Technologies as well as lots of FBS from other sources failed to support monolayer formation. This is not a different strategy compared with other laboratories in the CF research community who rely on medium supplements such as Ultraser G and PC-1 supplement for growth and monolayer formation in cell lines and primary cultures of CF and non-CF airway epithelial cells.

Transient Transfections of Nonpolarized Epithelial Cells

A few unifying methods must be stated. First, although manufacturers claim that these lipids are as efficient when used in serum-containing medium, we found that OptiMEM-I serum-free medium provided the best medium for lipofection with all lipids screened. Second, extensive washes with this medium before and after transfection also helped prepare cells for transfection and remove excess lipid, respectively. Third, less lipid than suggested in a manufacturer's instructions (usually based on transfection of heterologous cells) was also better for epithelial cells to limit toxicity and improve gene transfer. Fourth, growth of epithelial cells (and heterologous cells as well) on a Vitrogen-coated substrate prevented cells from detaching during washes before and after transfection. Fifth, epithelial cells were seeded into six-well plates and were transfected at 50-75% confluence 2 days after seeding as a standard practice for nonpolarized cells.

Per well of a six-well plate, the following protocol was performed for each lipid. Only the lipids that were effective in our study are shown here. Amounts of DNA, lipid, and medium were increased in proportion to surface area of culture.

LipofectAMINE PLUS. A 15-ml tube was used to mix 1 µg of cDNA and 6 µl of PLUS reagent in 150 µl of OptiMEM-I medium. In a separate 15-ml tube, 4-6 µl of LipofectAMINE reagent were mixed with 150 µl of OptiMEM-I medium. Tthe two tubes were incubated separately at room temperature for 15 min. The contents of the LipofectAMINE reagent tube were then transferred and mixed with the cDNA and PLUS reagent. The single tube containing both lipids and the cDNA was incubated at room temperature for another 15 min. During the two 15-min incubations, the cells were washed three times with OptiMEM-I medium. The volume of the mixture was diluted to 1.5 ml with OptiMEM-I, and the contents of the tube were added to the well of the cells.

LipofectAMINE 2000. A 15-ml tube was used to mix 1 µg of cDNA in 150 µl of OptiMEM-I medium. In a separate 15-ml tube, 3-4 µl of LipofectAMINE 2000 reagent were mixed with 150 µl of OptiMEM-I medium. The two tubes were incubated separately at room temperature for 15 min. The contents of the LipofectAMINE 2000 reagent tube were then transferred and mixed with the cDNA. The single tube containing LipofectAMINE 2000 and cDNA was incubated at room temperature for another 15 min. During the two 15-min incubations, the cells were washed three times with OptiMEM-I medium. The volume of the mixture was diluted to 1.5 ml with OptiMEM-I, and the contents of the tube were added to the well of the cells.

Effectene. A 15-ml tube was used to mix 1 µg of cDNA and 6 µl of Enhancer lipid in 150 µl of OptiMEM-I medium. The tube was incubated at room temperature for 10 min. To the same tube, 12 µl of Effectene lipid were added to the mixture containing cDNA and Enhancer lipid. The single tube containing both lipids and the cDNA was incubated at room temperature for another 10 min. During the two 15-min incubations, the cells were washed three times with OptiMEM-I medium. The volume of the mixture was diluted to 1.5 ml with OptiMEM-I, and the contents of the tube were added to the well of the cells.

FuGENE 6.0. A 15-ml tube was used to mix 1 µg of cDNA and 6 µl of FuGENE 6.0 reagent in 150 µl of OptiMEM-I medium. The tube was incubated at room temperature for 15 min. During the 15-min incubation, the cells were washed three times with OptiMEM-I medium. The volume of the mixture was diluted to 1.5 ml with OptiMEM-I, and the contents of the tube were added to the well of the cells.

Cells were viewed 24-48 h later for GFP fluorescence and were harvested at 48 h for LUC reporter gene activity or for CFTR immunoprecipitation.

Transient Transfections of Epithelial Monolayers

Epithelial cells were seeded onto Vitrogen-coated Transwell filter supports as described above. After 48 h, RTE was measured with a Voltohmmeter (Millipore). MDCK monolayers required daily RTE measurement and feeding, whereas airway epithelial monolayers were measured and fed every 2 days. Monolayers were transiently transfected 3-5 days after seeding. Routinely, RTE was still improving and had not plateaued fully during this period. Before transfection with lipid reagents and mammalian expression vectors, RTE was measured and monolayers were washed three times with OptiMEM-I medium (Life Technologies). Monolayers were kept in this medium while transfection cocktails were prepared. Morphology was performed to determine that these epithelial cells did, in fact, form monolayers with ZO-1 staining of tight junctions (data not shown). All transfection cocktails were prepared in OptiMEM-1 medium in the absence of FBS for consistency and to prevent FBS neutralization of DNA:lipid complexes. Per 6-mm-diameter monolayer, 0.5 µg of mammalian expression vector bearing LUC (pRL-CMV; Promega, Madison, WI), GFP (pGL-1 Green Lantern; Life Technologies or pEGFP-C1; Clontech), and/or CFTR cDNA [pRSV-CFTR, a generous gift of Garry Cutting, Johns Hopkins Univ., Baltimore, MD; pGFP-CFTR, a generous gift of Bruce Stanton, Dartmouth, Hanover, NH; or pcDNA3.1 WT-CFTR, Univ. Alabama at Birmingham (UAB) CF Center] was transfected into the monolayer. Again, amounts of DNA, lipid, and medium were increased in proportion to surface area of filter support.

The following lipid reagents were screened: Lipofectin, LipofectAMINE, LipofectAMINE PLUS, LipofectAMINE 2000, and DMRIE-C (Life Technologies); FuGENE 6.0 (Roche Diagnostics); Tfx-10, Tfx-20, and Tfx-50 (Promega); and Effectene (Qiagen). Per 6-mm-diameter monolayer, 6 µl of each lipid reagent were used (with the exception of Effectene, where 12 µl were added per monolayer). This 6-µl amount also applies to the Enhancer lipid reagent for Effectene and the PLUS reagent for LipofectAMINE PLUS. Lipid:DNA complexes mixed as a cocktail were added to both sides of the monolayer (1-ml total volume; 0.5 ml to apical and basolateral sides of the monolayer), except where sidedness of lipid-mediated gene transfer into the monolayer was tested. Transfection cocktails were mixed according to the manufacturer's instructions; however, OptiMEM-I was used as the medium for making the mixtures in all cases. Monolayers were incubated for 6 h at 37°C in a humidified, 5% CO2 incubator. RTE was measured before and after transfection. Routinely, RTE dropped 100-200 Omega  · cm2 during the 6-h transfection; however, after feeding with FBS-containing medium and an overnight recovery, the monolayer RTE returned to or exceeded pretransfection values (see RESULTS). The chemistry of the lipid mixtures is known for Lipofectin, LipofectAMINE, and DMRIE-C. Unfortunately, these lipids failed to transduce monolayers well. The information is proprietary for the other reagents, which, interestingly enough, were the most efficacious.

LUC and GFP Reporter Assays

LUC enzyme reporter assays were performed using the Dual Luciferase Reporter Assay System (Promega). The medium was removed from the filters, and the filters were washed with Ca2+,Mg2+-free PBS. Passive lysis buffer (100 µl of 1× buffer) was added to each well and allowed to incubate for a minimum of 15 min. After 15 min, lysis was achieved when the cells were loosened, removed, and broken apart by vigorous pipetting. The lysate was then placed in an Eppendorf tube. Twenty microliters of lysate were removed and placed in a luminometer tube containing 50 µl of of Luciferase Assay Reagent II and measured in the luminometer to provide a zero value (reagent only works for firefly LUC). Stop and Glo Reagent (50 µl) was then added (reagent is the substrate for Renilla LUC, whose cDNA is carried by the pRL-CMV vector), luminometer readings in triplicate were taken, and an average was calculated to determine LUC activity. Fresh reagents were used for entire sets of samples to ensure equivalent readings across all transfected samples.

GFP-transfected monolayers (grown always on clear polyester filters to visualize GFP fluorescence in the monolayer) were visualized every 24 h following transfection using a Nikon Eclipse TE-200 inverted microscope equipped with GFP epifluorescence. The relative transfection efficiency was approximated visually and was confirmed by a second investigator. Digital images were taken using a Nikon Eclipse TS100 microscope equipped with a digital camera port. A Nikon CoolPix E990 digital camera was attached to the camera port via an MDC CoolPix lens. Live cells were imaged in standard culture medium 48 h posttransfection; no fixation procedures were needed. 16HBE14o- cell monolayer images were taken at ×10 magnification to maximize the field of cells imaged, whereas IB3-1 CF cells were imaged at ×20 magnification.

Voltohmmeter Open-Circuit and Ussing Chamber Short-Circuit Current Measurements of Monolayer Electrical Properties

With the use of the Millipore MilliCell ERS Voltohmmeter that uses chopstick electrodes with Ag-AgCl pellets, RTE of the monolayers formed on the 6.5-mm filters were measured to assess the relative maturation of the monolayers and to monitor RTE before and after transient transfection. As a screen for CFTR chloride channel activity in a polarized epithelium under unstirred and noncirculated conditions in serum-containing medium, RTE was measured initially under basal conditions, followed by addition of 50 µM amiloride to the apical side of the monolayer to inhibit sodium channels. Addition of amiloride either produced no change in RTE or caused an increase in RTE due to the closing of basally active sodium channels. In the presence of apical amiloride, forskolin (2 µM) was added to the apical and basolateral sides of the monolayer, resulting in a decrease in RTE due to forskolin-mediated activation of CFTR chloride channels.

RANTES Chemokine ELISA

This assay, done in the absence or presence of transiently transfected CFTR and with or without induction with the cytokines TNF-alpha and IFN-gamma , has been described in detail previously (5) and was performed 48 h after transient transfection on samples of the medium harvested from apical and basolateral sides of the transfected monolayer. RANTES is only transcribed, translated, and secreted when wild-type CFTR is expressed via transient or stable transfection into CF epithelial cell models devoid of functional or detectable CFTR protein (5). Thus secretion of the chemokines, RANTES, is an indirect end point showing successful transduction and expression of CFTR.

Immunoprecipitation of CFTR

Two days after transient transfection of polarized monolayers, the monolayers were washed for 10 min three times with PBS. The monolayers were kept at 4°C during the washes. The monolayers were then lysed in 150 mM NaCl, 50 mM Tris, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS (pH 8.0) containing protease inhibitors. Monolayers were cut out of the support while in RIPA buffer, and the monolayers were incubated in RIPA buffer on ice for an additional 30 min to complete the lysis. The samples were then vortexed vigorously and were subjected to centrifugation at maximum speed in an Eppendorf Microcentrifuge at 4°C for 20 min. The supernatant was transferred to a new tube. The protein concentration was measured using a BCA kit and a microplate reader. CFTR anti-NBD-1 antibody and protein A agarose were then added to immunoprecipitate CFTR on a rotator shaker at 4°C for 2 h minimum or overnight. After the immunoprecipitation incubation, the samples were again subjected to centrifugation at maximum speed at 14K at 4°C for 2 min. The supernatant was discarded, and the pellet of beads was kept. The pellet was washed twice in RIPA buffer and once in PKA buffer. Catalytic subunit of cAMP-dependent protein kinase (PKA) and [gamma -32P]ATP were added to phosphorylate the CFTR regulatory domain to enhance its visualization by phosphorimaging. Samples were centrifuged at maximum speed for 2 min at room temperature and washed three times in RIPA buffer. Samples were mixed with 2× sample buffer and incubated at 37°C for 10-15 min. Samples were run on a 6% Tris glycine gel at 150 V for 90 min, dried on a gel dryer, and analyzed on a PhosphorImager.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Efficacy of Transient Transfection of Epithelial and Heterologous Cells with LipofectAMINE PLUS, LipofectAMINE 2000, FuGENE 6.0, and Effectene

Our initial results with transient transfection of heterologous cells such as COS-7 and HEK-293 cells and CF human airway epithelial cells like IB3-1 led us to optimize conditions for nonpolarized human airway epithelial cell cultures. We used both GFP and LUC reporter genes to quantify the percentage of cells positively transfected and the total amount of protein being synthesized from the introduced vector, respectively. Such an analysis is shown in Table 1 for 16HBE14o- cells grown in nonpolarized culture on Vitrogen-coated plates. Effectene was toxic to these cells and to IB3-1 cells, whereas FuGENE 6.0 was only marginally effective (data not shown). In Table 1, data are shown for LipofectAMINE PLUS and LipofectAMINE 2000 for GFP visualization and for LUC activity from protein lysates. LipofectAMINE PLUS, when used in 4- or 6-µl volumes of both the PLUS priming lipid and the LipofectAMINE transfecting lipid, transduced 40-50% of 16HBE14o- cells by GFP fluorescence, had the most robust LUC signal and had no toxic effect. When added at 8-µl or greater volumes, these lipids began to show toxicity. With LipofectAMINE 2000, 3 µl of this newly available lipid transduced 60% of 16HBE14o- cells, had the most robust LUC signal on any condition assessed in Table 1, and had no toxic effect. Greater amounts of LipofectAMINE 2000 began to be toxic. Together, these results show that LipofectAMINE PLUS and LipofectAMINE 2000 are efficient at promoting gene transfer in human airway epithelial cells.

                              
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Table 1.   Efficacy of lipid-mediated transient transfection in non-polarized 16HBE14o- cells transfected at subconfluence

IB3-1 cells were transfected transiently under nonpolarized conditions to visualize and image GFP to show that the majority of transfected cells were GFP positive under the most optimal conditions (Fig. 1). Again, normalizing for the surface area of a well of a six-well plate, 3 µl of LipofectAMINE 2000 or 4 or 6 µl of both LipofectAMINE and PLUS reagent (in 1.5 ml of OptiMEM-I medium volume) were optimal in transfecting this CF human airway epithelial cell line without toxicity. A conservative estimate showed that at least 50% of the cells were GFP positive when transfected with 6 µl of both LipofectAMINE and PLUS reagent (Fig. 1A). Together, these data show that at least 50% of the cells in the culture are transfected positively with GFP, that the LUC protein signal is robust in the lysates, and that LipofectAMINE PLUS, when used in a limited amount, is not toxic to epithelial cells grown under nonpolarized conditions.


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Fig. 1.   Images of green fluorescent protein (GFP) reporter protein fluorescence in live, nonpolarized IB3-1 cystic fibrosis (CF) human bronchial epithelial cells to assess the percentage of positively transfected cells in the culture and immunoprecipitation of cystic fibrosis transmembrance conductance regulator (CFTR) protein from a transiently transfected IB3-1 CF cell culture. A: IB3-1 cells imaged under GFP fluorescence (a) and phase microscopy (b). One microgram of EGFP-C1 vector and 6 µl of both LipofectAMINE and PLUS reagent were used for this culture. B: duplicate transfections were performed on separate days, and cells were prepared for immunoprecipitation. "Band C" refers to the fully processed and fully glycosylated form of CFTR, whereas "Band B" refers to the immature, core glycosylated form of CFTR that is retained in the endoplasmic reticulum (ER) or an intermediate ER-associated compartment. No butyrate induction, chemical chaperones, or proteasomal inhibitors were used in these experiments. WT, wild-type CFTR; Delta F, Delta F508 CFTR mutation; EV, empty vector control (pcDNA 3.1 plasmid without CFTR gene). Note the lack of any endogenous CFTR protein in this IB3-1 system.

Transfer of reporter genes is promising and shows efficacy; however, these gene cassettes are small. It is possible that smaller size vectors may overestimate transfection efficiency, because they gain entry to cells more easily. Thus we also assessed whether vectors bearing the CFTR gene gain as efficient entry into nonpolarized IB3-1 CF cells that are null for biochemically or functionally detectable CFTR. Figure 1B shows two representative immunoprecipitation experiments where vectors bearing the wild-type form of CFTR and the most common disease-associated CFTR mutation, Delta F508, were transfected transiently into IB3-1 CF cell cultures with LipofectAMINE PLUS. Whereas empty vector-transfected cultures had no forms of CFTR protein (see Fig. 1B), wild-type CFTR showed the fully glycosylated "band C" form of CFTR protein, with a hint of the partially glycosylated "band B" form of CFTR. In contrast, Delta F508-CFTR, an ER retention mutant because of a folding defect in NBD-1, showed only the immature "band B" form of CFTR. Together, these data show that 1) IB3-1 cells are an ideal CFTR-null human airway epithelial expression system for CFTR expression studies, and 2) a strong biochemical signal for CFTR is achieved upon transient transfection with LipofectAMINE PLUS, indicative of efficient and robust gene transfer.

Optimization of Monolayer Formation in Non-CF and CF Airway Epithelial Cell Lines

Before applying this system to polarized epithelial monolayers, optimization of monolayer formation was necessary. For non-CF and CF airway epithelial monolayers, screened lots of FBS (see MATERIALS AND METHODS), which enhanced monolayer maturation and augmented RTE in monolayers, were used. MDCK epithelial cells were also used in this study, because they are the gold standard for epithelial cell monolayer biology. MDCK monolayer formation was independent of the FBS used; MDCK monolayers were grown in MEM containing 10% FBS. Keratinocyte growth factor (KGF; otherwise known as fibroblast growth factor-7, or FGF-7) was also tested for its effects on airway epithelial monolayer maturation, because KGF has been shown to affect lung epithelial growth and differentiation (8). Four conditions were compared: 1) 10% FBS, 2) 10% FBS plus 50 ng/ml KGF, 3) 20% FBS, and 4) 20% FBS plus KGF. For 16HBE14o- non-CF monolayers and IB3-1 CF monolayers, KGF (50 ng/ml) improved RTE when added to 10% FBS-supplemented medium. Further improvement was observed in RTE with MEM supplemented with 20% FBS (Table 2). No significant improvement was seen with KGF added to 20% FBS; however, the resistance did improve slightly. For CFT-1 monolayers, RTE values were independent of the amount of FBS (data not shown). However, addition of KGF to the medium increased RTE significantly, but only at the peak RTE measurement on day 3. Also shown in Table 2, monolayer maturation of the three airway epithelial models as well as MDCK epithelial cells was compared on clear polyester Transwell filter supports vs. nonclear polycarbonate Transwell filter supports coated with Vitrogen. In all airway epithelial models, monolayer development (measured as RTE) was enhanced on clear polyester filter supports (Table 2). In MDCK monolayer studies, polyester filters were better 24 h after seeding; however, polycarbonate filters were a better substrate for RTE maturation at later time points (Table 2).

                              
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Table 2.   Optimization of monolayer-forming culture conditions for airway and heterologous epithelial cells

Transient Transfection of Epithelial Cell Lines Grown as Monolayers with LUC Reporter Genes

Having optimized conditions with which to grow polarized epithelial monolayers, we screened a large panel of lipid transfection reagents for their ability to transduce 16HBE14o- monolayers with the LUC reporter gene. Initially, we were concerned that addition of lipid transfecting reagents in serum-free medium for a 6-h incubation would reduce RTE in a sustained manner.

Figure 2 shows representative data sets for three different efficacious lipid reagents on 16HBE14o- monolayers. Surprisingly, no lipid transfecting reagent had a sustained effect on RTE; only a transient and short-lived drop in RTE was observed that was reversed upon feeding and overnight incubation in standard culture medium (Fig. 2). RTE was measured for each set of monolayers studied throughout the project, as shown in Table 2 and before and after transient transfection. This data set is large; thus only typical examples are shown. Because this transient reduction was seen with every lipid reagent tested, we hypothesized that the OptiMEM-1 medium is the cause of the transient dip in RTE, which resolves when the monolayers are washed and fed with standard culture medium. This hypothesis was supported by an experiment performed in Fig. 7.


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Fig. 2.   Lack of effect on lipid transfecting reagents on transepithelial resistance (RTE) development in 16HBE14o- non-CF airway epithelial monolayers. In these experiments, monolayers were transfected transiently with LipofectAMINE PLUS (A), FuGENE 6.0 (B), or Effectene (C) and pRL-CMV-LUC. RTE was measured 2 days after seeding, before and after transfection on day 3, and each day (D) thereafter. Monolayers were harvested 48, 72, and 96 h later to perform LUC assays on the lysates (see Fig. 3). These are 3 examples of effects on transient transfection with different lipids on 12 monolayers. This procedure was performed for every monolayer transiently transfected in this study.

Because it was known that lipid reagents were not toxic to epithelial monolayers (even some lipids that were toxic to nonpolarized epithelial cells), LUC was transfected transiently into 16HBE14o- monolayers, and LUC activity was measured in monolayer lysates 48, 72, and 96 h after transfection. Lipofectin and LipofectAMINE had little efficacy in 16HBE14o- monolayers (Fig. 3A). In sharp contrast, LipofectAMINE PLUS transduced 16HBE14o- monolayers with LUC significantly at all three time points (Fig. 3A). DMRIE-C reagent had little effect, as did Tfx-20 and Tfx-50. Because DMRIE reagent was mixed with DOPE reagent in the study by Snyder (6), we also mixed DMRIE-C reagent with all of the lipid reagents listed in Fig. 3. DMRIE had no effect on the ability of any other lipid reagent to transfect 16HBE14o- monolayers (data not shown). In addition to LipofectAMINE PLUS, Effectene also transduced 16HBE14o- monolayers significantly at the 48- and 72-h time points (Fig. 3A). In a second series of experiments, similar results were observed for LipofectAMINE PLUS and Effectene (Fig. 3B). However, in addition to these reagents, LipofectAMINE 2000 and FuGENE 6.0, two additional proprietary lipid transfecting reagents that became available during this project, were also efficient at transducing 16HBE14o- monolayers with LUC (Fig. 3B). Of interest, combinations of two different lipid reagents had no additive effect on LUC gene transfer. Together, more newly developed lipid transfection reagents, LipofectAMINE PLUS, LipofectAMINE 2000, Effectene, and FuGENE 6.0, transfected 16HBE14o- monolayers transiently.


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Fig. 3.   Transient transfection of epithelial cell lines grown as monolayers with lipid transfecting reagents and Luciferase (LUC) reporter gene. In these experiments performed in triplicate, 16HBE14o- monolayers were transfected transiently with a subset of lipid reagents (A). Monolayers were harvested 48, 72, and 96 h later to perform LUC assays on the lysates. B: single lipid reagents or combinations of reagents were used for transducing 16HBE14o- monolayers with LUC, and lysates were harvested and examined for LUC activity only at the 48-h time point (again, 3 monolayers per condition). C: experiments were performed in triplicate on Madin-Darby canine kidney (MDCK) cell monolayers at the 48-h time point on 6.5-mm-diameter (left) and 12-mm-diameter filters (right). D: experiments were performed in triplicate on CFT-1 CF human tracheal epithelial cell monolayers at the 48-, 72-, and 96-h time points on 6.5-mm-diameter filters. E: experiments were performed on 16HBE14o- monolayers in triplicate to assess the sidedness of effective lipid transfecting reagents in LUC reporter gene transfer. Ap, apical; Bl, basolateral. Six 6.5-mm-diameter monolayers were tested for each condition. With LipofectAMINE 2000 and Effectene, the signal was significantly enhanced when complexes were added to both sides vs. apical only; however, the signal was not observed with the other lipids. We hypothesize that the OptiMEM-1 medium actually may loosen tight junctions (as reflected in the transient decrease in monolayer resistance) to allow some enhanced transduction.

Figure 3C shows a similar analysis of transient transfection of MDCK monolayers. In contrast to the studies of 16HBE14o- monolayers, only Effectene reagent transfected MDCK monolayers transiently and effectively in an initial series of experiments on 6-mm-diameter filter supports (Fig. 3C, left). LipofectAMINE PLUS produced a lower LUC signal. A second series performed on MDCK monolayers grown on 12-mm-diameter filters showed that Effectene, LipofectAMINE 2000, FuGENE 6.0, and, to a lesser extent, LipofectAMINE PLUS transfected transiently and effectively MDCK monolayers (Fig. 3C, right). Together with data for the 16HBE14o- monolayers, these data show that the above-mentioned lipid transfecting reagents transfect epithelial monolayers transiently. The heterogeneity of the LipofectAMINE PLUS data suggests that surface area of the monolayer may be an important factor in designing transient transfection experiments. This only came to light in MDCK monolayer experiments, because non-CF and CF human airway epithelial cell lines failed to form monolayers on filter supports larger than 6.5 mm in diameter.

To study CFTR in a polarized system that is truly null for CFTR functionally, we needed to 1) grow CF human airway epithelial cells as polarized monolayers (see Table 2) and 2) transiently transfect the monolayers with CFTR constructs. To assess the feasibility of transiently transfecting CF airway epithelial monolayers with lipid:DNA complexes, LUC transfer into CFT-1 monolayers (Fig. 3D) and IB3-1 monolayers (not shown) was performed in a manner similar to that for MDCK and 16HBE14o- monolayers. As with the above-mentioned systems, Effectene, LipofectAMINE PLUS, and FuGENE 6.0 were all effective in transduction.

Transient Transfection of Airway Epithelial Monolayers Occurs from the Apical Side of the Monolayer

Until this point, transfection cocktail was added to both sides of the monolayer to determine whether transient transfection of polarized epithelial monolayers was feasible. Once the most effective lipids were identified, we wanted to determine whether lipid reagents allow transfer of LUC mammalian expression vectors across the apical membrane, the basolateral membrane, or both. For this experiment, the most effective lipid transfecting reagents were tested on 16HBE14o- monolayers. Figure 3E shows the results for apical transfer, basolateral transfer, or apical and basolateral transfer. For each lipid transfecting reagent, addition of the lipid:DNA complexes to the apical side of the epithelial monolayer produced a LUC signal similar to that observed when complexes were added to both sides of the epithelium (Fig. 3E). Only a very weak signal not different from the "DNA only" control was observed in samples where complexes were added only to the basolateral side of the monolayer (Fig. 3E). These data show that effective lipid transfecting reagents introduce DNA into polarized epithelial monolayers across the apical membrane. Only a slight enhancement of the signal was noted when lipid:DNA complexes were added to both sides of the epithelium, suggesting that some loosening of the epithelium (as documented by the transient dip in RTE) may allow some additional gene transfer through the basolateral membrane. Lack of basolateral membrane transfer may be due simply to the lipid:DNA complexes being impeded by the collagen-coated filter support. It was difficult, if not impossible, to modify the support to try to enhance basolateral gene transfer without affecting the monolayer RTE.

Transient Transfection of Non-CF and CF Epithelial Cell Primary Cultures Grown as Monolayers with LUC Reporter Genes

It was only feasible to screen for the best conditions for transient transfection of polarized epithelial cell monolayers in terms of the best timing for gene and protein expression and the best lipid transfecting reagents in immortalized cell lines. However, similar efficacy may not hold for primary cultures of human airway epithelial cells grown on filter supports. To address this question, non-CF and CF human airway epithelial cell monolayers grown in primary cultures were transiently transfected with the best four lipid transfecting reagents in parallel with 16HBE14o- epithelial cell monolayers under similar conditions. In the context of these additional experiments, we also assessed the effect of growing 16HBE14o- cells in air:fluid interface culture vs. fluid:fluid culture, because the primary airway epithelial cell monolayers required air:fluid interface culture to polarize. We also examined combining LipofectAMINE PLUS and LipofectAMINE 2000 as well as using twice as much lipid transfecting reagent to facilitate. Figure 4A shows that transduction with LUC was, in fact, better in 16HBE14o- cells grown in air:fluid interface culture for FuGENE 6.0, Effectene, LipofectAMINE PLUS, and LipofectAMINE 2000. Moreover, 2× lipid made no difference for Effectene, whereas it impaired transduction with LipofectAMINE PLUS and LipofectAMINE 2000. If one compares the LUC activity recovered in these monolayers vs. that recovered earlier during optimization, our results with 16HBE14o- monolayers improved during the course of the study as well as upon inclusion of LipofectAMINE 2000 into our work.


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Fig. 4.   Transient transfection of epithelial cell primary cultures grown as monolayers with lipid transfecting reagents and LUC reporter gene. A: experiments were performed on 16HBE14o- monolayers in parallel to primary cell monolayers to assess the effect of air:fluid (Air:F) interface culture vs. fluid:fluid (F:F) culture as well as lipid combination cocktails (n = 4 per condition). Of course, during the course of the transient transfection, fluid containing lipid:DNA complexes was added to both sides of the epithelium for the 6-h transfection period. After several washes, the monolayer was subjected again to air:fluid interface culture, whereas fluid:fluid cultured monolayers never deviated from this condition. B: non-CF primary human small airway epithelial (NHSAE; n = 4 per condition) and sinus epithelial (NHSE; n = 1 per condition) cell monolayers were grown in air:fluid interface culture and transfected transiently. C: CF primary human nasal polyp (CFNP) epithelial cell monolayers were also tested (n = 4 per condition).

Figure 4, B and C, shows results from transient transfection of non-CF and CF human primary airway epithelial monolayers done in parallel with the 16HBE14o- monolayers grown in air:fluid interface culture in Fig. 4A. Primary cultures of non-CF human small airway epithelial cells (NHSAE) and non-CF human sinus epithelial cells (NHSE) were grown as monolayers in air:fluid interface culture and were transiently transfected with LUC (Fig. 4B), while primary cultures of CF human nasal polyp epithelial cells grown as monolayers were also examined (Fig. 4C). As with 16HBE14o- monolayers, non-CF primary monolayers were transduced best with LipofectAMINE 2000, and using a combination of LipofectAMINE PLUS and LipofectAMINE 2000 gave no added benefit or, actually, inhibited transduction. Interestingly, and in contrast, monolayers of CF human nasal polyp epithelial cells were transduced by both LipofectAMINE PLUS and LipofectAMINE 2000 individually; however, these primary CF monolayers were transduced best by a combination of LipofectAMINE PLUS and LipofectAMINE 2000.

Transient Transfection of Epithelial Monolayers with GFP As a Reporter

To assess approximately the percentage surface area transfected in an epithelial monolayer, transient transfection of GFP was performed in 16HBE14o- monolayers grown on a translucent polyester 6.5-mm-diameter filter. In general, our estimate, from multiple observers assessing the monolayers, was that 20-30% of the 16HBE14o- monolayer surface area was transduced with GFP using LipofectAMINE 2000, LipofectAMINE PLUS, or combinations of these effective lipids. LipofectAMINE 2000 was the most efficient, transducing 20-30% of the surface area on a consistent basis. The GFP fluorescence was less bright than the images show for nonpolarized IB3-1 CF cells. It was our intent to show an image of the entire surface area of a polarized 16HBE14o- monolayer grown on 6.5-mm-diameter translucent polyester filter support transiently transfected with GFP using LipofectAMINE 2000 and LipofectAMINE PLUS. However, these filter supports are not translucent enough when imaged on either GFP fluorescence microscope available to our laboratory. This was likely due to the narrow filter and plastic filter support that also contributed significant autofluorescence. Because GFP images could not be obtained on our best monolayers, we used CFTR gene transfer as an functional and biochemical end point (see Figs. 6-8).

However, we could assess GFP gene transfer in 16HBE14o- cells grown packed together as a very confluent, pseudopolarized monolayer (Fig. 5, in contrast to subconfluent cells studied in Table 1) on a larger filter support. It should be noted that the RTE was significantly less on the 24-mm filter supports (200-300 Omega  · cm2) than on the 6.5-mm filter supports (600-1200 Omega  · cm2). LipofectAMINE 2000 was the best lipid transfecting reagent and showed little toxicity. A collage of GFP fluorescent images with increasing amounts of LipofectAMINE 2000 shows that 6 µl of this lipid gave maximal GFP reporter gene transfer (Fig. 5). Again, a conservative estimate shows that ~20-30% of the surface area of the monolayer showed GFP fluorescence in a given field. For these monolayers, LipofectAMINE PLUS was not as efficient (only 10% of cells were transfected with 6 and 8 µl of both lipids); enhanced GFP (EGFP)-only and lipid-only controls showed no GFP fluorescence (data not shown). In contrast to nonpolarized CF and non-CF airway epithelial cells where LipofectAMINE PLUS was the most useful lipid transfecting reagent, these results show that LipofectAMINE 2000 was the most efficient lipid transfecting reagent as the epithelial cells pack together and become polarized. We did not attempt to add greater amounts of LipofectAMINE 2000 to achieve greater GFP-positive surface area; however, this may be possible without encountering toxicity.


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Fig. 5.   GFP gene transfer with LipofectAMINE 2000 in 16HBE14o- cell monolayers. A collage of images of GFP fluorescence of 16HBE14o- cell monolayers, taken 48 h after transfection, shows efficacy of 1 through 6 µl of LipofectAMINE 2000 (C-H; phase images are shown to left of each condition). One microgram of EGFP-C1 vector was used in each condition. DNA-only (A) and lipid-only (B) controls are shown. Of note, in Table 1, data for LUC, %GFP-positive cells, and toxicity were generated on 16HBE14o- cell cultures that were transfected at subconfluence and processed 48 h later at 80-90% confluence (e.g., never reached confluence). In the studies summarized in Table 1, LipofectAMINE PLUS was as efficient as LipofectAMINE 2000; however, LipofectAMINE PLUS was not as efficacious in 16HBE14o- cell monolayers grown on 24-mm supports.

Chloride Channel Function of CFTR in Transiently Transfected Monolayers

The ultimate goal of this work was to develop a polarized epithelial transient transfection system in which epithelium-specific proteins could be studied transiently in a setting that most approximated a polarized epithelial cell in vitro. Because our ultimate goal is to use this system to study CFTR biochemistry, cell biology, and physiology, Fig. 6 shows an assay for cAMP-activated CFTR chloride channel activity under open-circuit conditions measuring changes in RTE as a function of CFTR activity. CFBE41o- human CF airway epithelial cell monolayers were transfected at day 5 and studied on day 7. RTE was 1,532 ± 150 Omega  · cm2 (n = 12) at day 5, dropped to 523 ± 14 after the 6-h transfection period, and returned to a value of 2,209 ± 128 at day 6. On day 7, basal RTE was 2,713 ± 147 Omega  · cm2 (n = 12). In the presence of amiloride to block sodium channels (which caused only a modest increase in RTE; data not shown), forskolin-induced decrease in RTE was measured as an end point of CFTR chloride channel activity in the apical membrane. In CFBE41o- CF human bronchial epithelial cell monolayers, little change in RTE was observed after forskolin treatment in monolayers untransfected or mock transfected with empty vector (Fig. 6). Transient transfection of WT-CFTR into CFBE41o- monolayers homozygous for the Delta F508 CFTR mutation conferred a marked forskolin-induced decrease in RTE, suggesting that transient transfection of the polarized monolayer complemented the CF epithelium with a normal copy of the CFTR gene (Fig. 6). The real values were 2,954 ± 128 Omega  · cm2 (n = 6) before forskolin stimulation and 2,070 ± 153 Omega  · cm2 (n = 6) after stimulation. As expected, Delta F508-CFTR did not complement CFBE41o- monolayers with a functional CFTR (Fig. 6). Similar data were generated in CFT-1 monolayers; however, the overall RTE and changes in RTE were smaller in magnitude. Together, these data suggest that transient transfection of epithelial monolayers is sufficient to observe the electrical activity of the CFTR chloride channel in polarized epithelial monolayers. Further optimization of monolayer maturation in these and other CF epithelial cell models is necessary to develop higher resistance monolayers that display a significant transepithelial voltage to do meaningful Ussing chamber experiments.


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Fig. 6.   CFTR chloride channel function in the apical membrane of transiently transfected epithelial monolayers assessed under open-circuit conditions. After RTE measurements under basal conditions and after amiloride treatment of the apical side of the monolayers for 10 min, forskolin was added to both sides of the epithelium for a 10-min incubation. The decrease in RTE is a measure of cAMP-activated CFTR chloride channels or chloride channels positively regulated by CFTR. n = 6 for WT-CFTR-transfected monolayers (pRSV-WT) and n = 2 for untreated, empty vector-transfected, and Delta F508-CFTR-transfected monolayers (pRSV-Delta F508). Absolute RTE values are provided in text. Similar data were generated in CFT-1 monolayers; however, the overall RTE and changes in RTE were smaller in magnitude.

CFTR-Dependent Rescue of RANTES Expression in Transiently Transfected Monolayers

Further proof of positive CFTR transduction of polarized airway epithelial cells was generated by assessing CFTR-dependent rescue of RANTES gene expression and secretion. Previously, in nonpolarized CF epithelial cell systems, we showed that CF airway epithelial cells fail to express the chemokine RANTES at the mRNA or protein level (5). To address CFTR-regulated gene expression in polarized airway epithelia indirectly by measuring RANTES secretion as the end point, CFT-1 epithelial monolayers were transfected transiently with Effectene reagent. ELISA analysis of the apical and basolateral medium showed that CFTR alone enhanced RANTES expression and secretion across the apical membrane (Fig. 7). Addition of the cytokines TNF-alpha and IFN-gamma (20 ng/ml each) enhanced RANTES expression and secretion across the apical and basolateral membrane, but only in the presence of CFTR (Fig. 7). No RANTES expression or secretion was observed in the absence of CFTR (Fig. 7). Together, these data agreed with previous studies using transient transfection with LipofectAMINE PLUS in nonpolarized cells (5). Importantly, this was the first study in which CFTR rescue of RANTES expression (or the expression of any gene) was accomplished by transient transfection of an epithelial monolayer. Rescued RANTES release into the apical and basolateral medium is also consistent with its role in modulating the activity of immune cells resident in the airway as well as chemoattraction of specific populations of immune cells from the interstitium and bloodstream.


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Fig. 7.   CFTR-dependent rescue of RANTES expression in transiently transfected CFT-1 CF human airway epithelial monolayers. Experiments were performed in triplicate by sampling apical and basolateral medium 48 h after transfection of WT-CFTR or empty vector (mock) and after overnight treatment with the cytokines TNF-alpha and IFN-gamma (20 ng/ml each) or with carrier alone to both sides of the monolayer. All samples were transfected with pRL-CMV-LUC to standardize for transfection efficiency. Effectene was used as the transfecting lipid reagent for these experiments. RANTES ELISA was performed, and data were normalized to LUC activity measured after monolayers were lysed, after medium aliquots were harvested.

Immunoprecipitation of CFTR protein from transiently transfected monolayers. Ultimately, an important utility of this work is to transfect airway or heterologous epithelial monolayers transiently to study the cell biology and biochemistry of the CFTR protein in its normal physiological setting, the apical membrane of a polarized epithelium. Figure 8 includes representative SDS-PAGE gels of CFTR immunoprecipitation from IB3-1 and CFT-1 CF human airway epithelial monolayers transiently transfected with several different lipid transfecting reagents or combinations of lipid transfecting reagents. In IB3-1 monolayers, LipofectAMINE PLUS and LipofectAMINE 2000 alone or together with Effectene transfected monolayers effectively so that CFTR protein could be detected by immunoprecipitation (Fig. 8). Effectene and FuGENE 6.0 alone or in combination failed to transduce IB3-1 monolayers efficiently enough to observe a CFTR protein signal. In CFT-1 cells, Effectene alone failed to yield a CFTR protein signal; however, LipofectAMINE PLUS and LipofectAMINE 2000 in combination with Effectene produced a CFTR immunoprecipitation signal (Fig. 8). Together, these data show that transient transfection of resistive CF human airway epithelial monolayers is feasible and is an ideal system to study CFTR protein cell biology and biochemistry in a polarized epithelium.


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Fig. 8.   Detection of transiently transfected CFTR protein via immunoprecipitation of CFTR from CF human airway epithelial monolayer lysates. A: transiently transfected IB3-1 monolayers were harvested 48 h afterollowing transfection with the different lipid cocktails listed (empty vector was pEGFP-C1 without the CFTR cDNA; the CFTR vector used was pGFP-WT-CFTR). Lane A, T84 monolayers included as a positive control; lane B, empty vector with Effectene reagent; lane C, CFTR with Effectene reagent; lane D, CFTR with Effectene and FuGene 6.0; lane E, FuGene 6.0; lane F, CFTR with Effectene and LipofectAMINE PLUS; lane G, CFTR with LipofectAMINE PLUS; lane H, CFTR with Effectene and LipofectAMINE 2000; and lane I, CFTR with LipofectAMINE 2000. B: transiently transfected CFT-1 monolayers were harvested 48 h after transfection with the different lipid cocktails listed (empty vector was pEGFP-C1 without the CFTR cDNA; the CFTR vector used was pGFP-WT-CFTR). Lane A, T84 monolayers as a positive control; lane B, empty vector with Effectene reagent; lane C, CFTR with Effectene; lane D, CFTR with Effectene and LipofectAMINE PLUS, and lane E, CFTR with Effectene and LipofectAMINE 2000. A hint of less core glycosylated B band is seen in the T84-positive control and in the positively transfected samples (but, interestingly, not in mock controls or unsuccessful transfections). These data are representative of 3 such experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

These studies are the first to show effective transient transfer of reporter genes and CFTR into human primary or immortalized non-CF and CF airway epithelial cells or MDCK epithelial cells grown as polarized epithelial monolayers. Very few studies have documented such findings (3, 6, 9) in polarized epithelial cell systems. This work benefited from the optimization of transient transfection in nonpolarized epithelial cell systems that supported our previous work with CFTR biochemistry, cell biology, and physiology. With this polarized monolayer system, we can ask many questions about CFTR cell biology and protein biochemistry, CFTR-dependent signaling and effects on gene expression, and CFTR physiology in a native and more in vivo-like polarized system. Early work suggests that CFTR behaves very differently in its cell biology and function in human airway epithelial cells vs. heterologous cells (Bebok Z and Collawn JF, unpublished observations).

With the exception of LipofectAMINE PLUS and LipofectAMINE 2000, however, many lipids were toxic to epithelial and heterologous cells grown in nonpolarized conditions. In particular, Effectene was very toxic to nonpolarized epithelial cells. Why then is a lipid like Effectene not toxic to polarized epithelial monolayers? The simplest explanation is that the epithelial cells, when packed tightly together as a resistive monolayer, are more resistant to toxicity. At no time in any transient transfection of a polarized epithelial monolayer did we have a large, sustained decrement in RTE that did not recover. In fact, the transient decrease in RTE caused by the lipid-mediated transfection was due to 6-h exposure to the serum-free medium rather than the DNA:lipid complexes.

Although it was not the intent of these studies to discover a new vehicle of gene transfer for CF, we may have, in fact, stumbled onto some lead candidates for lipids that transduce human airway epithelial cells from the apical side. Experiments examining the sidedness of LipofectAMINE reagent-based gene transfer showed that the side of entry was apical. Lack of basolaterally mediated transfer may have been caused by the collagen-coated filter support itself; however, the success of apically mediated delivery has led us to begin experiments with instillation of these more newly developed lipid transfecting reagents in mice.

It is important to emphasize that the same efficacy with a given lipid reagent in these epithelial cell systems may not hold for an epithelial cell from a different tissue. A screen of a panel of lipid reagents is necessary to select the reagent that is most effective for a given epithelial model. The four reagents shown to be effective in this study would be a blueprint from which to begin such an analysis. For example, early work with T84 and HT-29 monolayers suggests that LipofectAMINE 2000 is the best lipid for transient transfection of these human intestinal epithelial systems, whereas the others are less efficacious (Schwiebert EM, unpublished observations). In contrast, CFPAC-1 monolayers preferred LipofectAMINE PLUS over the other lipids (Zsembery A and Schwiebert EM, unpublished observations). Nevertheless, there are even newer lipid reagents that have come to market since we began this study, and these should be tested. However, because we were successful with mammalian expression vectors bearing LUC and GFP as well as multiple vectors bearing the CFTR cDNA, we feel that this system could be applied to most mammalian expression vector constructs as large as 10 kb.

In conclusion, transient transfection of polarized epithelial monolayers is feasible and effective. It is hoped that investigators will use these foundations to apply this system to epitheliim-specific cDNAs and proteins of interest to study the cell biology and physiology of epithelium-derived proteins in nonpolarized epithelial cells and, ultimately, in a polarized epithelium, their native and natural environment. Future studies are needed to also use this novel technology to assess whether these lipid transfecting reagents are suitable for gene transfer for CF in vivo.


    ACKNOWLEDGEMENTS

We thank Peter Snyder at the University of Iowa for helpful initial discussions concerning experiences with transient transfection of polarized epithelial monolayers. We thank the University of Alabama at Birmingham (UAB) CF Center for providing CFBE41o- monolayers for this study. The bulk of the non-CF and CF human primary cultures were provided by N. A. McCarty; however, a limited number of non-CF sinus epithelial primary cell monolayers were also provided by the UAB CF Center. N. A. McCarty thanks B. J. Duke (Emory University) for assistance with establishing primary cell cultures from patient samples. E. M. Schwiebert thanks A. Zsembery, Bill Rice, and Liz Hanson for help with LUC reporter gene assays.


    FOOTNOTES

These studies were funded by National Institutes of Health (NIH) Grants R01-DK-54367 and R01-HL-63934 (to E. M. Schwiebert), a CF Foundation Research Grant (to L. M. Schwiebert), and a CF Foundation Research Grant and NIH Grant R01-DK-60065 (to J. F. Collawn). J. F. Collawn, L. M. Schwiebert, and E. M. Schwiebert should be considered co-senior authors of this study, because this was a long-lived collaboration between their three laboratories.

Address for reprint requests and other correspondence: E. M. Schwiebert, Dept. of Physiology and Biophysics, Univ. of Alabama at Birmingham, MCLM 740, 1918 Univ. Blvd., Birmingham, AL 35294-0005 (E-mail: eschwiebert{at}physiology.uab.edu); or L. M. Schwiebert, Dept. of Physiology and Biophysics, University of Alabama at Birmingham, MCLM 966, 1918 University Blvd., Birmingham, AL 35294-0005 (E-mail: lschwieb{at}uab.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published November 6, 2002;10.1152/ajpcell.00435.2002

Received 20 September 2002; accepted in final form 28 October 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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