1Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, and 2Division of Cancer Research, National Health Research Institutes, Taipei 100, Taiwan, Republic of China; and 3Department of Anatomy and Cardiovascular Research Institute, University of California, San Francisco, California 94143
Submitted 19 February 2003 ; accepted in final form 3 May 2003
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ABSTRACT |
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fluorescence-activated cell sorting; green fluorescent protein; Ras small GTPases; anoikis
As a first step to employ the power of these gene expression systems, a plasmid encoding the regulatory protein must be transfected into the target cell line. To select a stable clone possessing the most desirable characteristic, this process frequently involves intensive cloning, cell culturing, and testing of each candidate clone and is very time consuming. Because of the effort demanded for screening for useful clones by the conventional strategy, large-scale screening cannot be performed easily, and this makes a successful outcome uncertain. Therefore, extensive application of inducible gene expression systems, although desirable for studying gene functions, has been limited.
Independent control of two target genes by two inducible gene expression systems would be invaluable in many applications. Although such an idea has been tested and successfully executed (7, 16, 17, 27), more general application has been hindered by the technical limitations of setting up inducible cells as described above. Here we present a simple and efficient fluorescence-activated cell sorter (FACS)-based scheme to generate inducible expression cells without going through the tedious single clone selection procedure. With this selection strategy, a population of highly inducible cells is enriched in which ecdysone- and tetracycline-controlled expression systems function together to facilitate dissection of Rho family GTPase signaling pathway's involvement in regulating anoikis.
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MATERIALS AND METHODS |
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Cell culture and transfection. Madin-Darby canine kidney (MDCK)
cells and human embryonic kidney 293 (HEK-293) cells were grown in DMEM
containing 10% fetal bovine serum at 37°C in a humidified atmosphere
containing 5% CO2. Transfection was performed using Lipofectamine
2000 (Invitrogen) according to the manufacturer's instructions. For luciferase
reporter assay, one million MDCK or HEK-293 cells were transfected with 3.6
µg of pIND-Luc or pTRE-Luc and 0.4 µg of pRL-TK (Promega), a
Renilla luciferase-expressing construct driven by thymidine kinase
promoter as an internal control to normalize the transfection efficiency. One
million MDCK or HEK-293 cells were transfected with 4 µg of the fluorescent
reporter plasmids pIND-GFP and pTRE-GFP. To obtain enough cellular lysates for
protein two-dimensional gel electrophoresis, we used 16 µg of pU-MRac1-V12
and 16 µg of pIND-IB
(DN) to transfect 10 million cells for
each experiment. To enrich ecdysone-regulated clones, 20 million MDCK and
HEK-293 cells were transfected with 80 µg of pVgRXR and selected in the
presence of 300 or 150 µg/ml zeocin, respectively. Tetracycline-inducible
cells were established by selecting pM2-IRES-Puro and pTet-TS (at a ratio of
1:15)-transfected cells in 2.5 µg/ml puromycin.
Flow cytometry. One to ten million cells were sorted by Vantage flow cytometer (Becton-Dickinson). The fluorescence profiles were analyzed and processed using CellQuest v3.3 software.
Fluorescence microscopic examination, photographic presentation, and immunoblotting. Detailed experimental procedures were performed as described previously (10, 11).
Luciferase and LacZ assays. One million MDCK or HEK-293 cells were transfected with the reporter constructs as described in Cell culture and transfection. The cells were trypsinized 6 h after transfection and plated onto 24-well plates with 1 µg/ml doxycycline (Sigma) for the tetracycline-inducible system or 5 µM ponasterone (Stratagene) for the ecdysone-inducible system added to half of the wells to activate the reporter. Inducers were absent from the other half of the wells as noninduced controls. The cells were incubated for 40 h before being harvested for dual luciferase assays (dual firefly and Renilla luciferase reporter assay; Packard BioScience). Promoter activities were measured in a Wallac's luminometer (1420 multilabel counter) and expressed by dividing the firefly luciferase activity with Renilla luciferase activity; each value was the mean of a triplicate measurement. Every assay was repeated at least three times. When pIND-LacZ was included in the reporter assay, its activity was evaluated using the Galactostar kit (Tropix).
Cell death ELISA assay. MDCK cells were induced to express transgenes for 24 h as an attached culture. They were subsequently trypsinized and cultured in suspension on ultralow-attachment plates (Costar) at a density of 5 x 104 cells/ml for 16 h before being processed for measurement of DNA-histone complex by using the Cell Death ELISA kit (Roche Molecular Biomedicals).
Two-dimensional gel electrophoresis. Cells were extracted in 7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 1% 1,4-dithioerythritol (DTE), and 40 mM Tris for 1 h at room temperature and centrifuged at 200,000 g for 1 h at 22°C, and protein was normalized by Bradford assay before being loaded into Immobiline DryStrip (pH 310NL, 18 cm; Pharmacia). The strips were rehydrated with 100 µg of cell lysate in 8 M urea, 2% CHAPS, 2% immobilized pH gradient (IPG) buffer (pH 310; Pharmacia), 2 mM tributylphosphine, and trace bromphenol blue for 46 h at 0 V and 12 h at 50 V. Isoelectric focusing was performed at 20°C by using the IPGphor IEF system (Pharmacia) at 300 V for 3 h, a gradient of 3003,500 V for 3 h, 3,500 V for 3 h, and constant 5,000 V. After the focusing was completed after 125,000 Vh, strips were incubated in equilibration buffer (50 mM Tris, pH 6.8, 6 M urea, 2% SDS, and 30% glycerol), first with 2% DTE for 15 min and then with 2.5% iodoacetamide and bromphenol blue for another 15 min. Equilibrated strips were inserted onto 916% gradient SDS-PAGE gels and run at 40 mA for 1 h, 50 mA for 3 h per gel at 12°C in Protean IIxi Multi-Cell (Bio-Rad). Analytical gels were silver stained and preserved by air drying between sheets of moistened cellophane.
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RESULTS |
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Establishing ecdysone-inducible lines in MDCK and HEK-293 cells by mass cell culture and cell sorting based on a fluorescent reporter. To illustrate the effectiveness of the proposed strategy, we report the application of this strategy to generate an ecdysone-inducible expression system in an MDCK cell line. Twenty million MDCK cells were transfected with pVgRXR. About 500 transfectants survived selection of zeocin and were pooled together. This candidate pool was then transiently transfected with pIND-GFP, a GFP reporter plasmid constructed under the control of an ecdysone-responsive element, and divided into two pools immediately after transfection. One pool of the cells was kept in medium to which 5 µM ponasterone, a synthetic ecdysone analog, was added, whereas the other pool was maintained as noninduced control. Two days later, cells were subjected to FACS. The difference in fluorescence profiles under induced and noninduced conditions revealed a window for optimal responders to be positively sorted (Fig. 1A). These selected cells were then expanded in the absence of ponasterone until enough cells had been grown for another round of positive selection. This strategy was very effective in enriching clones that apparently had the characteristics of ecdysone-inducible expression of GFP reporter. However, as the percentage of inducible cells increased after each round of positive selection, progressively more cells were found in the selected pool that possessed unregulated expression characteristics. To eliminate these leaky responders, cells after the fourth round of positive selection were expanded and transfected again with pIND-GFP and left in medium without ponasterone for negative selection. We noted that there were two populations of cells: the first with fluorescence even under noninduced conditions and the second with no fluorescence (S+4 cells in Fig. 1A). To better appreciate the dynamic change of ecdysone inducibility in this cohort of cells, we retained aliquots of cells before each round of positive FACS selection and after final negative selection, and we transfected these cells with pIND-GFP at the same time for direct observation under a fluorescence microscope (Fig. 1B). FACS was so effective in purging the leaky clones that only a few cells still possessed leaky expression characteristics after a single round of negative selection (S+4-1 cells in Fig. 1B).
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To quantitatively demonstrate the efficiency of this inducible GFP reporter and FACS-based selection strategy, we transfected pIND-Luc into aliquots of MDCK cells collected before and after each round of selection. The candidate clones capable of ecdysone inducibility were enriched after each round of positive selection as demonstrated by gradually increased luciferase activity and activation factor after each round of positive selection (Fig. 2A). Although the absolute induced luciferase activity seemed to decrease after one round of negative selection, the relative induction factor actually did not change significantly.
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We also applied a similar selection strategy to sort HEK-293 cells that had been stably transfected with pVgRXR. The selection was so efficient, presumably resulting from the transfection-friendly nature of HEK-293 cells, that just two rounds of positive selection permitted successful collection of a pool of cells (293.Ec) possessing an impressive degree of ecdysone-inducible expression (Fig. 2B).
A tetracycline-regulated activator and silencer system potentiates selection of inducible cell lines based on a fluorescent reporter. Because the FACS-based selection was very efficient in establishing the ecdysone-inducible expression system, we tried to apply a similar strategy to set up a tetracycline-inducible expression system in 293.Ec, which was selected for ecdysone-regulated expression. When pTRE-GFP, a GFP reporter plasmid constructed under tetracycline-responsive promoter, was transfected into HEK-293 cells stably expressing tTA (293.Ec.1), the fluorescence profiles under induced and noninduced conditions were so similar that it was hard to identify a window to positively select the inducible cells by FACS (Fig. 3A). That window became even more inconspicuous when pTRE-GFP was transfected into another population of HEK-293 cells that stably expressed rtTA (293.Ec.2) (Fig. 3A). Such a dilemma is not related to the fact that ecdysone-inducible system has been established in these particular HEK-293 strains, because similar fluorescence profiles were also observed when pTRE-GFP was transfected into HEK-293 cell strains that do not express ecdysone receptor proteins (data not shown). The main reason for this failure is the transient status of transfected GFP plasmids. Under such conditions, the reporter plasmids are usually multicopied per cell and exist in an episomal form, which precludes the chromatin repression effect and therefore elicits gene expression by the interaction between endogenous transcriptional machinery and the minimal CMV promoter element in the tetracycline-regulated promoter (1, 4).
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To solve this problem, we adopted the tetracycline-controlled
activation/repression system that had been demonstrated to reduce unwanted
background expression of tetracycline promoter-regulated transgene
(3,
4,
22). By fusing KRAB silencing
domains to TetR, a tetracycline-controlled silencer (tTS) is generated that
binds to the operator sequence within the tetracycline-regulated promoter and
actively shields the promoter from transcriptional activation in the absence
of tetracycline. Addition of tetracycline releases tTS from the promoter and
at the same time activates the promoter via rtTA
(4). Furthermore, we adopted an
engineered version of rtTA, rtTA-M2, in this transactivator/transrepressor
system because rtTA-M2, which possesses five amino acid substitutions (S12G,
E19G, A56P, D148E, and H179R) of rtTA, had been proved to display a
considerably lower background activity and higher range of induction than rtTA
(13,
25). The HEK-293 line, which
had been installed with the ecdysone-inducible expression system (293.Ec;
Fig. 2), was transfected with
pM2-IRES-Puro and pTet-TS, followed by puromycin selection. Initial assay of
the selected cells (293.Ec.3) confirmed the efficiency of the
tetracycline-controlled activation/repression system for reducing background
activity of transiently transfected pTRE-GFP
(Fig. 3A). We then
processed 293.Ec.3 for two rounds of positive selections and two rounds of
negative selection using the FACS strategy described above and designated the
resulting cells as 293.Ec.3.+2-2. Luciferase activity assay showed that the
reporter gene could be efficiently regulated by doxycycline in this cell line;
the inducibility of the reporter was up to two orders of magnitudes
(Fig. 3B).
Furthermore, its expression was independent of the expression status of the
-galactosidase reporter gene controlled by the ecdysone-inducible system
(Fig. 3B). Similarly,
the expression of ecdysone promoter-driven
-galactosidase reporter was
also independent of the status of tetracycline system-controlled luciferase
reporter (Fig.
3C).
A tetracycline and ecdysone double-inducible system assists delineation of signaling hierarchy of Ras-related small GTPases involved in regulating anoikis. To demonstrate the usefulness of a double-regulated expression system in dissecting complex signaling pathways within a cellular context, we set up a stable MDCK cell line capable of inducible expression of two related small GTPases, Cdc42 and Rac1, under ecdysone- and tetracycline-regulated expression systems, respectively. We first introduced the ecdysone-inducible system, using the FACS-based selection strategy described, into a Rac1 dominant negative mutant (Rac1N17)-expressing MDCK cell line that had been demonstrated to express the mutant GTPase tightly and efficiently under a tetracycline-repressible system (10, 11). We named this population of ecdysone-inducible cells Rac1N17.1. A constitutively active Cdc42 mutant (Cdc42V12)-expressing plasmid was then constructed under the control of the ecdysone-inducible promoter. This plasmid was stably transfected into the Rac1N17.1 to generate a MDCK clone in which myc-tagged Rac1N17 and FLAG-tagged Cdc42V12 mutants could be tightly, efficiently, and independently expressed under tetracycline- and ecdysone-regulated expression systems, respectively (Fig. 4A). Rac1 and Cdc42 both belong to the RhoA family of the Ras superfamily of GTPases. Cdc42 has been demonstrated to act upstream of Rac1, and Rac1 upstream of RhoA, in controlling actin cytoskeleton organization in fibroblasts (19). To examine whether the same signaling cascade also plays a role in anoikis (5), a detachment-induced apoptotic process, we took advantage of the Rac1N17 and Cdc42V12 double-expressing cell line and observed how differential expression of these two transgenes affected apoptosis of MDCK cells kept in suspension condition. Figure 4B shows expression of Rac1N17-enhanced anoikis of MDCK cells, as demonstrated previously (2), whereas Cdc42V12 inhibited anoikis of MDCK cells, and expression of both mutants resulted in an enhancing effect on anoikis. This finding implies that Rac1 acts downstream to Cdc42 along the signaling pathway governing detachment-induced apoptosis in MDCK cells.
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A double-inducible system facilitates identification of signaling
molecules relaying complex signaling pathways by proteomic assays.
Besides its effect on actin cytoskeleton organization, the Rac1 small GTPase
also regulates gene expression through its effect on transcriptional factors
(26). Rac1 has been shown to
regulate NF-B activity in response to physiological and pathological
stimuli such as mechanical stretch and hypoxic stress
(12,
20,
24). Although Rac1 has been
demonstrated to activate MEKK1, which activates JNK, and JNK in turn activates
NF-
B by stimulating degradation of I
B
(15), there might be other
unknown pathways linking Rac1 and I
B. Taking advantage of the ecdysone
and tetracycline double-regulated 293.Ec.3.+2-2 cell line, we transiently
expressed constitutively active Rac1 and dominant negative I
B genes and
explored the possibility of using this double-inducible system to dissect the
signaling pathways linking Rac1 and I
B. We transfected constitutively
active Rac1 gene to 293.Ec.3.+2-2 under the control of tetracycline-regulated
promoter (pU-MRac1-V12). By Western blot, we demonstrated that this
myc epitope-tagged Rac1 mutant could be tightly regulated by
doxycycline (Fig. 5A).
In the mean time, a truncated mutant missing the first 70 amino acids of
I
B
was cloned downstream to the ecdysone-responsive promoter to
make pIND-I
B
(DN). Deletion of the NH2-terminal 70
amino acids of I
B removes the potential phosphorylation sites at serine
32 and 36 positions by I
B kinase and has been demonstrated to inhibit
subsequent activation of endogenous NF-
B in a dominant negative fashion
(personal communication, Yen-Shen Lu). The expression of this transgene was
robustly induced with the addition of 5 µM of ponasterone, whereas the
transgene was not detected under noninduced conditions
(Fig. 5B). We obtained
cellular lysates for two-dimensional gel electrophoresis under three different
conditions: 1) neither Rac1V12 nor I
B(DN) was expressed;
2) only Rac1V12 was expressed; and 3) both Rac1V12 and
I
B(DN) were expressed. After comparing protein expression profiles for
each condition, we could detect several types of changes in expression pattern
[Fig. 5, CF;
please also refer to the Supplemental
Material1 for this
article (published online at the American Journal of Physiology-Cell
Physiology web site) to view whole images of the silver-stained gels].
There were spots on silver-stained two-dimensional gels that were not visible
under condition 1 but that became visible under conditions 2
and 3 (Fig.
5C). In contrast, there were spots that appeared only
under condition 2 (Fig.
5D). The former likely represent Rac1 downstream genes
that are not activated through NF-
B, whereas the latter represent Rac1
downstream genes that are dependent on NF-
B activation. In addition, we
also detected spots with changes in relative intensity or distribution, and
these possibly represent proteins whose posttranslational modification status
are affected by the activities of Rac1 and NF-
B
(Fig. 5, E and
F). These findings disclose latent candidates along Rac1
signaling pathways and exemplify the potential of applying this
double-regulated expression system in proteomic studies to dissect complex
signaling pathways.
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DISCUSSION |
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Although regulated gene expression system is such a goal for cell
biologists, it is by no means easily attainable using an individual cell
cloning strategy. Besides the conventional screening strategy using luciferase
assay, there are alternative strategies such as screening clones with
integrated tetracycline-regulated transactivator gene by PCR amplification of
the DNA extracted from the candidates or screening them by using tTA- or
rtTA-specific antibodies. Although these methods offer alternative options for
luciferase assay, these strategies still rely on individual cell cloning and
are therefore very time and effort demanding. Furthermore, positive signals
from either a PCR or Western blot result could poorly correlate with the
inducibility of the selected clones. In fact, strong expression of regulatory
proteins such as tTA or rtTA seems to be negatively selected because they may
squelch endogenous transcriptional factors
(8). The traditional strategy
of selecting optimal responder is very demanding, in that it could cost an
experienced researcher 6 mo to assessing 230 clones before finally
identifying an ideal responder (personal communication, Y. Altschuler, Hebrew
University of Jerusalem). In contrast, our new strategy routinely screens
5001,000 candidate clones collectively at the same time, thanks to the
conjugated use of inducible GFP reporters and FACS. On average, each round of
positive or negative selection takes 710 days, depending on the
recovery percentage of sorted cells, and the full time course for enriching a
useful population of inducible cells takes just a few months. This advantage
certainly enhances the possibility of selecting tightly regulable and highly
inducible clones. In addition, we have not observed any drifting of the
induction/suppression phenotypes of the double-regulated MDCK and HEK-293 cell
lines after more than 20 passages, which demonstrates the long-term
applicability of this system.
During preparation of this article, we noted that a similar FACS selection strategy had been proposed for generating an inducible expression system (22, 23). Whereas tetracycline- and ecdysone-inducible systems could be established efficiently in retrovirus-transduced cells, the retrovirus-based GFP reporters are stably integrated in the genome of the established cell lines and may interfere with some application, e.g., subcellular localization by immunofluorescence. Although the inducible GFP reporters used in our FACS-based strategy might also integrate into the genome of some host cells after repeated transient transfections, there were still cells possessing inducible expression characteristics without having the GFP reporter integrated in their chromosomes (data not shown). Furthermore, the major difference between our work and the retroviral approach resides in our attempt to extend this FACS-based strategy to set up a tetracycline-inducible system and final success in combining ecdysone- and tetracycline-inducible systems together for broader application.
Although we generated the ecdysone and tetracycline expression systems sequentially by FACS-based sorting of GFP fluorescent cells, it is theoretically possible to set up a double inducible system simultaneously. Substitution of one of the two inducible GFP reporters, pIND-GFP or pTRE-GFP, with construct expressing red fluorescence-emitting protein such as DsRed would permit establishment of ecdysone and tetracycline systems at the same time by two-color fluorescence sorting (14).
The combination of tetracycline-regulated and ecdysone-inducible expression
systems offers an experimental modality of inducing two genes of interest
independently in a temporal and dose-controlled manner. This system could be
combined with research tools with a molecular precision at a genomic scale,
such as DNA microarray or proteomic assay, to dissect complex signaling
pathways. Although thousands of genes could be analyzed simultaneously by
these global approaches, the candidates' number might be just too elusive for
meaningful target genes to be identified precisely. The advantage of two
independent inducible expression systems allows massively parallel studies of
the interaction of two candidate genes in a homogenous cellular environment.
Consider the example of Rac1 and NF-B for illustration of the potential
of this dual regulatory expression system. Rac1 is known to activate
NF-
B, but Rac1 does not directly link to NF-
B. To search for the
possible molecules connecting Rac1 to NF-
B along the pathway,
constitutively active Rac1 (Rac1V12) and dominant negative I
B
[I
B-(DN)] could be inducibly expressed independently using the system
described above. Conceivably, there are three groups of proteins (groups
X, Y, and Z) whose expression patterns could be affected by the
expression of Rac1V12. Assuming that group X directly relays the
signaling between Rac1 and NF-
B, group Y is indirectly
activated by Rac1 through the influence of NF-
B, whereas group
Z proteins are specifically affected by Rac1 but not related to the
activation of NF-
B. If we induce the expression of I
B-(DN) on
top of Rac1V12 expression, group Y proteins would be back to their
baseline expression pattern (as in Fig.
5D) and group Z proteins would be not affected
at all (as in Fig.
5C), whereas the expression of group X proteins
might be affected in a complicated pattern (as in
Fig. 5, E and
F). Therefore, interesting spots could be pursued by
sensitive assay such as microcapillary liquid chromatography followed by mass
spectrometric identification.
In summary, a dual regulatory expression system, when combined with global expression approaches such as DNA microarrays or proteomic assays, could be an invaluable research tool for signaling transduction studies.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
1 Supplemental material for this article may be found online at
http://ajpcell.physiology.org/cgi/content/full/00064.2003/DC1.
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