Construction and Application of an Inducible System for Homogenous Expression Levels in Bulk Cell Lines

Stringently controlled conditional expressing systems are crucial for the functional characterization of genes. Currently, screening of multiple clones to identify the tightly controlled ones is necessary but time-consuming. Here, we describe a system fusing Tet (tetracycline)-inducible elements, BAC (bacterial artificial chromosome) and Gateway technology together to allow tight control of gene expression in BAC-transfected eukaryotic bulk cell cultures. Recombinase cloning into the shuttle vector and the BAC facilitates vector construction. An EGFP (enhanced green fluorescent protein) allows FACS (fluorescence activated cell sorting) and the BAC technology ensures tight control of gene expression that is independent of the integrating site. In the current first application, our gene of interest encodes a β-catenin-ERα fusion protein. Tested by luciferase assay and western blotting, in HTB56 lung cancer cells the final BAC E11-IGR-β-catenin-ERα vector demonstrated sensitive inducibility by Tet or Dox (doxycycline) in a dose-dependent manner with low background, and the EGFP was an effective selection marker by FACS in bulk culture HTB56 and myeloblastic 32D cells. This is a highly efficient tool for the rapid generation of stringently controlled Tet-inducible systems in cell lines.


Introduction
The rapid development of genomic functional research requires stringently controlled expression systems. Tet (tetracycline)-regulated expression systems are widely used. Tet-inducible systems are divided into two classes: one is TetOff, which is constitutively active in the absence of Tet or Dox (doxycycline), whereas off in the presence of Tet or Dox [1,2]; the other one is TetOn, which becomes active only in the presence of Tet or Dox [3]. One of the advantages of the TetOn system is that, after addition of the inducer, the induction is more rapidly than with the TetOff system following depletion of Tet or Dox. Additionally, gene expression levels can be controlled by adding different doses of inducers [4].
However, it is often cumbersome to clone genes into BAC including two steps: first, clone a gene of interest into an intermediate shuttle vector by traditional restriction digestion and ligation; second, the gene of interest is recombined into BAC from the shuttle vector. In the case of multiple expression and functional analysis, proper restriction sites have to be selected according to the different sequences of genes in above-mentioned step I, and then step II is repeated. It is a time-and moneyconsuming process. However, this work could be simplified by Gateway Technology using lambda phage-based site-specific recombination instead of restriction endonuclease and ligase to insert a gene of interest into the shuttle vector. The DNA recombination sequences (attL, attR, attB, and attP) and the LR or BP Clonase enzyme mixtures that mediate the lambda recombination reactions are the foundation of Gateway Technology. In our current study, we cloned a versatile shuttle vector pIGR-RFC. To use this system, the gene of interest is cloned into an entry vector (pENTR), which is then mixed in vitro with pIGR-RFC and the Gateway Clonase enzyme mixtures. Site-specific recombination between the att sites (attR x attL) generates an expression clone and a by-product. Thus, the reading frame C (RFC) in the shuttle vector is replaced by the gene of interest [22]. In this way, different genes of interest can now easily be cloned into a universal shuttle vector and then into a BAC.
In our former utilization of Tet-controlled BAC in generating transgenic mice, it appeared that some strains have strong Tetinducible expression of gene, while others do not, indicating clonal variation of the Tet-controlling system. In order to avoid time-and money-consuming selection of dozens of clones, the enhanced green fluorescent protein (EGFP) may be used as a selection marker because of its convenience for fluorescence activated cell sorting (FACS). By the means of flow cytometry, positivetransfected cells can be easily purified. Thus, in cellular and animal experiments EGFP could act as a useful visible marker by the fluorescence in positive clones.
To the best of our knowledge, our current Tet-controlled expression system is the first combination of Tet-inducible elements, BAC and Gateway technology. It boasts the tight and quantitative expression inducibility by Tet or Dox, the gene cloning convenience by Gateway technology, stable expression in BAC vector and efficient selection for EGFP contributing to a universal technical tool for the analysis of gene expression and its function.  Figure 1A and C. Its DNA sequence is provided as supplementary material (Supporting Information S1).

BAC Transfection
HTB56 cells were seeded 8 hours before transfection into 6-well plates with a density of 300,000 cells per well. Transfection was performed with Lipofectamine transfection reagent (Invitrogen, Carlsbad, CA, USA) by using 1 mg of super-coiled BAC DNA per well. For luciferase assay, 0.1 mg of pRLSV40 encoding renilla luciferase was cotransfected with BAC as internal control to normalize the transfection efficiency. Tet or Dox (Sigma, St. Louis, MO, USA) was added into the culture medium 24 hours after transfection.
Before transfection into 32D cells, the supercoiled BAC DNA was linearized by digestion with I-SceI restriction enzyme (New England Biolabs, Ipswich, MA, USA). For electroporation, 3610 6 32D cells suspended in 300 ml culture medium were transferred into an electrocuvette with a diameter of 4 mm containing 32 mg linearized BAC DNA. The electrical conditions were at 300 V, 6 ms using Pulse Generator EPI 2500 (Fischer, Heidelberg, Germany).

Dual-Luciferase Activity Assay
Activities of the firefly luciferase and Renilla luciferase in a single sample were measured sequentially using the Dual-Luciferase Report Assay System (Promega, Madison, Wisconsin, USA). Briefly, after 24 hours of Tet or Dox treatment, cells were rinsed twice with phosphate-buffered saline (PBS) and then lysed in 120 ml of passive lysis buffer at room temperature for 15 min. 10 ml of the cell lysate were quickly mixed with 100 ml of Luciferase assay reagent in a luminometer tube. The light emission for the firefly luciferase was recorded for 10 s after a 5 s premeasurement delay using a TD-20/20 luminometer (Turner Designs Instrument, CA, USA). Subsequently, 100 ml of Stop&Glo reagent was added to the same tube to inactivate the firefly luciferase while activating the Renilla luciferase. Variation in transfection efficiency was normalized by dividing the value of the firefly luciferase activity with that of the Renilla luciferase activity.

Western Blot Analyses
For preparation of whole-cell lysates cells were washed with icecold PBS and lysed for 30 minutes on ice in RIPA buffer with 150 mM NaCl as described [24]. Cell lysates were cleared at 20 000 g for 10 minutes. After adjustment of protein concentrations, the lysates were boiled in SDS sample loading buffer for 5 minutes and separated by SDS-polyacrylamide gel electrophoresis (PAGE, 4-12%, Invitrogen). Gels were blotted on a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA) and stained with the anti-human b-catenin first antibody (0.38 mg/ml, BD Biosciences, NJ, USA). Antibody binding was detected with a horseradish peroxidase (HRP)-coupled secondary antibody followed by chemoluminescence detection (ECL Plus, Amersham Pharmacia, Uppsala, Sweden).

Statistics
Quantitative data are presented as means plus standard deviation (SD). Statistical analyses were performed with SPSS, version 10.0 (SPSS Science, Chicago, IL, USA). Statistical significances of overall differences between multiple groups were analyzed by one-way ANOVA analysis. P values of 0.05 or less were considered significant.

Results and Discussion
To clarify the functional roles of various candidate genes screened by microarray, we established a Tet-inducible expressing system. As shown in Figure 1, the system we are describing here consists of a pENTR vector, an intermediate vector pE11-IGR-RFC and BAC E11. The pE11-IGR-RFC vector accommodates all the elements required for Tet-regulated gene expression including two expression cassettes, one of which encodes the elements responsive to Tet or Dox, and the other one of which encodes the rtTA2 S -M2, an optimized rtTA construct exhibiting significantly increased sensitivity to Dox [25]. An internal ribosome entry site (IRES) is added between the rtTA2 S M2 and EGFP to allow expression of these two genes driven by a single hCMV promoter. The EGFP acts as a sorting marker by flow cytometry in eukaryotic cells. The Tet-responsive expression cassette contains a bidirectional promoter P tet bi, driving expression of two genes simultaneously. The firefly luciferase is a surrogate marker for the expression of the gene of interest, which can be conveniently recombined into Gateway RFC from a pENTR vector. In the current study, the gene of interest is the b-catenin-ERa fusion gene (Fig. 1B). The backbone of pE11-IGR-gene of interest also comprises two homologous arms HA-A and HA-B sites for recombination with BAC in special bacteria resulting in a destination BAC E11-IGR-gene of interest (Fig. 1D).
Then, tetracycline inducibility of BAC E11-IGR-gene of interest was evaluated. HTEB56 cells transiently transfected by E11-IGR-b-catenin-ERa were exposed to different doses of Tet, Dox or not. We assayed the activity of luciferase, a sensitive surrogate marker reflecting the expression of the gene of interest in the other direction of P tet bi. As shown in Figure 2A, there is a dose dependent induction of luciferase activity to 0.05,5 mg/ml Tet or 0.01,1 mg/ml Dox. The BAC vector is more sensitive to Dox for conditional expression regulated by tetracycline. The promoter P tet bi allows simultaneous bidirectional expression: in one direction is the reading frame C (FRC) of Gateway system, into which the gene of interest can be recombined from pENTR plasmid conveniently, and in the other direction is luciferase as a surrogate marker for the expression of the inserted gene of interest. The sequence of rtTA2 S -M2, a reverse transactivator of P tet bi driven by hCMV was also cloned into this shuttle vector. To avoid inserting a third expression cassette, an internal ribosome entry site (IRES) was added between the rtTA2 S M2 and the EGFP to allow translation and expression of these two proteins from a single promoter hCMV. The homologous arms HA-A and HA-B serve as recombination sites between pE11 and the BAC. than to Tet. When the concentration of Dox is increased to 1 mg/ ml, the luciferase activity is over 80 times higher than that in the absence of Tet and Dox (P,0.001). The dose-dependent inducibility indicated that the expression level of the gene of interest can be regulated deliberately by adding different doses of Dox. In this way, the expression of the gene of interest is controlled not only qualitatively but also quantitatively in transiently transfected bulk cultures.
Furthermore, the inducible expression of b-catenin-ERa was evaluated. In our current study, we used pENTR-b-catenin-ERa as a donor for in vitro recombination with Gateway RFC (Fig. 1B). The fusion gene b-catenin-ERa encodes a chimaeric protein comprising wild type b-catenin fused with hormone binding domain of estrogen receptor a. As a result, the DNA fragment of b-catenin-ERa is recombined into BAC. Western blotting was performed after treatment with the indicated concentrations of Dox or Tet for 24 hours. The molecular weight of b-catenin-ERa fusion protein and wild type b-catenin is 136 and 92 kD respectively. Our antibody to b-catenin recognized both bcatenin-ERa fusion protein and the endogenous wild type bcatenin which acted as loading control. In line with the results of the luciferase assay, the expression of b-catenin-ERa was induced by Dox in a dose-dependent manner. Also, Tet (5 mg/ml) induced significant expression of b-catenin-ERa with very low background in non-Tet exposed cells. Importantly, the very low background suggests very little leakiness of inducibility in our current system, which makes it possible to control the expression of the gene of interest in a very stringent mode.
As a selection marker, EGFP positive cells can be sorted by FACS after transfection. In the final vector BAC E11-IGR-bcatenin-ERa, the expression of EGFP is constitutive and independent of the Tet-regulated cassette. As shown in Figure 3, there are less than 10% green cells 48 hours after transfection, however, around 70% after sorting by flow cytometry. This result indicated that the EGFP is an effective marker for FACS selection. The relatively low transfection efficiency is likely due to the large size (over 100 kb) of the BAC vector. There have been reports of successful transfection of supercoiled BAC DNA into Hela cells with Effectene reagent (Qiagen, Valencia, CA) [26]. In HTB56 cells, we used lipofectamine reagent (Invitrogen). Although the transient transfection efficiency is not high, the low background and high inducibility guarantee usefulness of this BAC system for transient transfection studies. Also, we succeeded in electroporation of linearized (see materials and methods section) BAC E11-IGR-b-catenin-ERa into 32D cells. It is suggested that it is necessary to optimize the transfection conditions in view of cell line specificity. Another cause of the low percentage of green cells is that the final vector BAC E11-b-catenin-ERa is not able to replicate episomally. One of the reasons why the expression of some episomal replicating vectors are not stable over time is that the selection can not distinguish episomal and genomic integrated ones, and the episomal expression is not as stable as that of the integrated [25]. Considering expression stability, we did not design a eukaryotic Ori in the final vector BAC E11-gene of interest. The advantage is that in the stable cell lines we will get, our gene of interest has integrated into the genome compared to the analysis of multiple clones.
In summary, our current Tet-controlled BAC system has tight and dose-dependent inducibility especially to Dox. It is a potential tool to study the functional role of unknown genes in bulk, for example, genes that have been identified in a high-throughput and comprehensive manner.