Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

Summary

A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies.

Abstract

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 cells^2,3. 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 siRNAs^5-11. 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 centrosome^13,14 and apoptosis signaling^15,16. 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.

Protocol

1. Cloning of pcDNA6_TetR_IRES_blast

1. 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.
2. 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.
3. 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.
4. 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

1. 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.
2. 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.
3. 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.
4. 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.
5. Check cells daily, ensuring that all cells on the untransfected plates have died within the first week. Change selection media every 2-3 days.
6. 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.
7. 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.
8. 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.
9. For each clone to be tested, freeze one well of confluent cells and harvest cells in the other well for subsequent testing by immunoblotting.
10. 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.
11. 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

1. 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.
2. 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.
3. 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

1. 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.
2. On day 2, transfect one plate with 2 μg of pTER (or pT-Rex DEST30) using Fugene 6. Do not transfect the second plate.
3. On day 3, add standard growth medium containing 5 μg/ml blasticidin and 500 μg/ml zeocin (or 1 mg/ml G418).
4. 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.
5. 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.
6. 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.
7. 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.
8. 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.
9. 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.
10. 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.
11. 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.

Results

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...

Discussion

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 desir...

Materials

Name	Company	Catalog Number	Comments
Fetal Bovine Serum(FBS)	Invitrogen	16000-044	Tested Tet-free
Blasticidin	Invivogen	ant-bl-1
Zeocin	Invivogen	ant-zn-5
G418	PAA laboratories	P31-011	100 mg/ml in media
anti-TET02	MoBiTec GmbH	TET02	Use at 1/1000 to 1/2000 for WB
pcDNA6/TR	Invitrogen	V1025-20
pT-Rex DEST30	Invitrogen	12301-016
Tetracycline	Sigma	87128	2 mg/ml in ethanol
Doxycycline	Sigma	D9891	2 mg/ml in water
Cloning cylinders	Bellco Glass Inc.	2090-00808	re-useable