Gomez-Martinez, M., Schmitz, D., Hergovich, A. Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression. J. Vis. Exp. (73), e50171, doi:10.3791/50171 (2013).
A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon1, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.
Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.12 modified the shRNA expression vector pSUPER5 by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.
Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14 and apoptosis signaling15,16. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.
1. Cloning of pcDNA6_TetR_IRES_blast
- As illustrated in Figure 3, perform a partial digest of the pcDNA6/TR plasmid (V1025-20, Invitrogen) with the restriction enzymes XbaI and NcoI to remove the TetR gene and the promoter of the blasticidin resistance (BlastR) gene. Isolate the 4.6 kb vector fragment.
- To remove the polyadenylation sequence from the TetR gene, introduce by PCR an XhoI site immediately after the Stop codon of the TetR cDNA while conserving the XbaI site at the 5'end of the cDNA. Subclone the resulting fragment into pMigR117 using restriction enzymes XbaI and XhoI to generate the pMigR1_TetR plasmid.
- Digest the pMigR1_TetR vector with the restriction enzymes XbaI and NcoI to release the TetR_IRES fragment. Isolate the 1.25 kb insert fragment.
- Ligate the 4.6 kb pcDNA6/TR and the 1.25 kb TetR_IRES fragments. Transform the ligation product into competent E.coli and identify the desired pcDNA6_TetR_IRES_blast construct using standard molecular biology techniques.
2. Generation of Cell Lines Stably Expressing TetR
- On day 1, Seed 1x106 cells per 10-cm tissue culture plate using your standard growth medium (e.g. DMEM supplemented with 10% FCS). Seed two plates, one for transfection with pcDNA6_TetR_IRES_blast, and the other as a negative control for blasticidin selection. Ensure to use the lowest passage possible and the recommended growth medium.
- On day 2, transfect one plate with 2 μg of pcDNA6_TetR_IRES_blast using Fugene 6 (E2691, Promega) following the manufacturer's instructions. Do not transfect the second plate.
- On day 3, add standard growth medium containing 5 μg/ml blasticidin to both plates. Please note that the optimal selection concentration may vary for a given cell line and might need to be determined beforehand.
- On day 4, split the cells at 1/5, 1/25, 1/50 and 1/100 using 10-cm plates and standard growth medium containing 5 mg/ml blasticidin. Make sure to properly label plates containing untransfected or transfected cells. Prepare two plates per dilution step.
- Check cells daily, ensuring that all cells on the untransfected plates have died within the first week. Change selection media every 2-3 days.
- After 1-2 weeks, blasticidin-resistant colonies should begin to appear. Select plates containing 5 to 50 colonies to ensure that single colonies can be picked.
- When colonies have reached a diameter of approximately 5 mm (or bigger), isolate at least 12 independent clones using cloning cylinders to pick single colonies. Transfer each clone into a separate well of a 24-well tissue culture plate. When confluent, split the cells from each well into one single well of a 6-well tissue culture plate.
- Continue to culture clones in selection media. When confluent, split cells from each well into two single wells of a 6-well tissue culture plate.
- For each clone to be tested, freeze one well of confluent cells and harvest cells in the other well for subsequent testing by immunoblotting.
- Detect TetR by Western blotting using the mouse monoclonal anti-TET02 antibody (MoBiTec GmbH). Remember to include a lysate of the parental cell line as a negative control.
- Select 3 clones with the highest TetR expression and expand each clonal culture. Freeze stocks of each promising clone as soon as possible. Finally, examine the molecular and cell biological parameters of interest by comparing the parental cell lines with the TetR-expressing clones. Select the 'best' clone with the highest TetR expression that does not display any alterations in the molecular and cell biological parameters of interest.
3. Pilot Testing of pTER and pT-Rex DEST30 Vectors Expressing shRNA or cDNAs, Respectively
- Generate pTER plasmid with shRNA inserts as described12. Construct pT-Rex DEST30 vector (12301-016, Invitrogen) containing the cDNA of interest using Gateway technology (Invitrogen). In case of cDNA expression, addition of a tag to the cDNA will later facilitate detection of exogenously expressed protein.
- Transiently transfect the parental target cell line with pTER or pT-Rex DEST30 plasmids (expressing either shRNAs or cDNAs of interest) using the transfection reagent of choice. For testing of shRNA expression plasmids, ensure high transfection efficiencies can be achieved.
- Harvest transfected cells and process for immunoblotting using the appropriate antibodies, expecting to detect a depletion or overexpression of your gene of interest.
4. Generation of Stable Cell Lines with Tetracycline-inducible (Tet-on) Expression of shRNAs or cDNAs Using pTER or pT-Rex DEST30 Vectors
- On day 1, seed 1x106 cells of the 'best' TetR-expressing clone per 10-cm plate using your standard growth medium supplemented with certified Tetracycline-free serum (for example FBS from Invitrogen; 16000-014). Seed one plate for transfection with pTER (or pT-Rex DEST30), and one plate as negative control for double selection. Make sure to use the lowest passage possible.
- On day 2, transfect one plate with 2 μg of pTER (or pT-Rex DEST30) using Fugene 6. Do not transfect the second plate.
- On day 3, add standard growth medium containing 5 μg/ml blasticidin and 500 μg/ml zeocin (or 1 mg/ml G418).
- On day 4, split the cells at 1/5, 1/25, 1/50 and 1/100 using 10-cm plates and standard growth medium containing 5 μg/ml blasticidin and 500 μg/ml zeocin (or 1 mg/ml G418). Prepare two plates per dilution step.
- Check the cells daily, making sure that within the first week all cells on the untransfected plates have died. Change the standard growth medium containing blasticidin and zeocin (or G418) every 3-4 days.
- After 2-3 weeks, blasticidin/zeocin (or blasticidin/G418) double-resistant colonies should begin to appear. Select plates containing 5 to 50 colonies to pick single colonies.
- When colonies have reached a diameter of approximately 5 mm (or bigger), use cloning cylinders to pick single colonies. Isolate at least 24 individual clones. Transfer each clone into a separate well of a 24-well tissue culture plate.
- Culture clones in medium containing maintenance concentrations of blasticidin (5 μg/ml) and zeocin (250 μg/ml) [or G418 (0.5 mg/ml)]. When confluent, split the cells from each well into one single well of a 6-well tissue culture plate.
- For each clone to be tested, split one confluent well of a 6-well plate into three single wells of a 6-well plate. When confluent, freeze cells from one well. Use the remaining two wells for testing the tetracycline-inducible expression. Add tetracycline at a concentration of 1 μg/ml to one well, while leaving the second well tetracycline free.
- After 24 to 96 hr of incubation with Tetracycline, harvest cells and process for immunoblotting using appropriate antibodies. Shorter tetracycline incubation times are recommended when screening for the induction of cDNA expression, while longer tetracycline treatments will be necessary to determine shRNA-mediated depletion of endogenous proteins.
- Select at least two clones with the desired depletion/overexpression and expand each culture. Freeze stocks of each promising clone as soon as possible. When growing Tet-on clones for experiments always make sure to use media supplemented with Tetracycline-free serum and the respective selection antibiotics at maintenance concentrations.
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An example for the initial characterization of RPE-1 cell lines stably expressing TetR is shown in Figure 4. Note that all RPE-1 clones express varying levels of TetR (compare lanes 2 and 5), while the parental cell line (which serves as negative control) does not express the exogenous TetR protein (Figure 4, lane 1). This variation in TetR expression among RPE-1 Tet-on cell clones is expected, since the expression of the TetR expressing plasmids is highly dependent on the plasmid integration site.
The characterization of stable RPE-1 Tet-on cell clones expressing shRNAs directed against the MST3 kinase shows that several clones have to be tested in order to define the cell lines with the most efficient depletion of the gene of interest (Figure 5). In the illustrated case (Figure 5) cell clones #1, #2 and #3 were chosen for further analysis. The variation in depletion efficiency is expected, since the expression of shRNA expression is highly dependent on the plasmid integration site.
Figure 1. The differences between Tet-off and Tet-on expression systems are shown. The Tetracycline (Tet) repressor protein (TetR) has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system).
Figure 2. Description of the two consecutive manipulation steps necessary to generate human Tet-on cell lines with inducible expression of the shRNA or cDNA of interest. Step 1: Generation of cells stably expressing TetR. Target cells are transfected with the pcDNA_TetR_IRES_blast vector, and Blasticidin (Blast) resistant clones are selected. Several clones are picked and tested for TetR expression by immunoblotting. After testing for all molecular and cell biological parameters of interest (e.g. serum starvation response, cell proliferation rates, or expression of marker proteins), the 'best' TetR-positive clone is expanded and stored. Step 2: Generation of Tet-on cell lines with inducible shRNA/cDNA expression. The 'best' TetR-positive cells are transfected with pTER12 for shRNA expression or with pT-Rex DEST30 (12301-016, Invitrogen) for cDNA expression, and Blast/Zeocin or Blast/G418 resistant clones are selected, respectively. Several individual cell clones are picked, expanded and screened by immunoblotting for Tetracycline-inducible knockdown or expression of the gene of interest. Finally, clones of interest are expanded, re-tested and stored.
Figure 3. Generation of the pcDNA6_TetR_IRES_blast vector. The newly generated pMigR1_TetR plasmid was digested with XbaI and NcoI, and the released 1.25kb fragment containing the TetR and IRES (internal ribosome entry site) sequences was cloned into the displayed XbaI and NcoI sites of pcDNA6/TR (V1025-20, Invitrogen). The resulting pcDNA_TetR_IRES_blast vector expresses the TetR and the Blasticidin resistance gene (BlastR) using the same CMV promoter, thereby ensuring that all Blasticidin resistant cells also express TetR.
Figure 4. Analysis of RPE-1 cell clones stably transfected with pcDNA/TetR_IRES_blast. Parental RPE-1 cells were compared to RPE-1 cells stably expressing TetR by immunoblotting using anti-TetR (top panel) and anti-alpha-tubulin antibodies (bottom panel).
Figure 5. Analysis of RPE-1 TetR-positive clones stably transfected with pTER_shMST3. To identify cells with tetracycline-inducible expression of shRNAs targeting MST3, clones were grown in the absence (-) or presence (+) of Tetracycline (Tet) for 72 hr, before processing for immunoblotting using anti-MST3 (top panel) and anti-alpha-tubulin antibodies (bottom panel).
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We believe that Tet-on systems greatly facilitate the analysis of gene function, particularly in cell systems that are difficult to manipulate and/or when the manipulated gene is essential for cell survival. Furthermore, the ability to control gene expression by highly specific Tet-on systems offers the opportunity to study gene functions at different stages (for example during cell cycle progression or well-defined differentiation processes). The method presented here will enable researchers to generate the desired stable Tet-on cell lines within a reasonable time frame (normally 3-6 months, largely depending on the extent of molecular and cell biological profiling that is necessary for the characterization of TetR-positive cell lines). The successful generation and application of Tet-on cell lines, however, is critically dependent on several factors.
First, the selection of cells with sufficiently high expression of TetR is required to ensure stringent repression of transcription in the absence of tetracycline. Equally important, the initial characterization of the target cells expressing the TetR should cover all possible molecular and cell biological parameters that will be studied on the long term. In our experience, a well-characterized TetR-positive cell line is a very good starting point for numerous research projects.
Second, it is necessary to keep the tetracycline concentration as low as possible to prevent possible pleiotropic effects when testing and analyzing Tet-on clones for shRNA/cDNA expression. If possible, the tetracycline concentration should not be higher than 2 μg/ml (preferentially lower than 2 μg/ml). Moreover, since tetracycline has a half-life of about 24 hr in tissue cell culture medium, experiments which last several days should consider additional rounds of tetracycline addition. Doxycycline, a synthetic tetracycline alternative, can also be considered when cytotoxic levels of tetracycline are of concern.
Third, tests should be applied differently for cDNA and shRNA expression. While Tet-on cells expressing cDNAs will easily produce detectable amounts of proteins within 24 hr, the depletion of endogenous factors by shRNA-mediated knockdown can take much longer. Generally, we recommend to test clones expressing shRNAs only 96 hr after tetracycline addition, which will lead to decreased levels of your gene of interest even if the half life of your target is 24 hr. The example shown in Figure 5 represents an 'ideal' screening result for shRNA-mediated depletion, and modifications to the testing protocol might be required to define the optimal knock down conditions. Last, but not least, we would like to stress the fact that well established screening tools (qRT-PCR probes, antibodies) will greatly facilitate the identification of your desired cell clones.
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No conflicts of interest declared.
We thank all members of our laboratory for helpful discussions. We thank Joanna Lisztwan and Christina Gewinner for critical reading of the manuscript. This work was supported by the BBSRC grant BB/I021248/1 and the Wellcome Trust grant 090090/Z/09/Z.A.H. is a Wellcome Trust Research Career Development fellow at the UCL Cancer Institute.
|Fetal Bovine Serum(FBS)
||100 mg/ml in media
||Use at 1/1000 to 1/2000 for WB
||2 mg/ml in ethanol
||2 mg/ml in water
||Bellco Glass Inc.
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