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
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ABSTRACT |
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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
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INTRODUCTION |
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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 -,
-,
and
-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.
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MATERIALS AND METHODS |
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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
F508-CFTR mutation, whereas IB3-1 bears the
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
· cm2 for 6.5-mm filter
monolayers; 1,000-2,000
· cm2 for 12-mm filter
monolayers; 500-1,000
· 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
· 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-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 [ ![]() |
RESULTS |
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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
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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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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,
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,
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
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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 16HBE14oFigure 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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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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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 16HBE14oTransient 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
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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 16HBE14oHowever, 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
· cm2) than on the 6.5-mm filter
supports (600-1200
· 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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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
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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-
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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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