Melissa Handler and Renato V Iozzo

1. Introduction

The use of antisense technology has been applied to a number of diverse organisms, including Drosophila melanogaster, plants, and mammals. The purpose is to take advantage of the base-pair specificity of both RNA:RNA and RNA:DNA interactions, ultimately to block protein production. This technique requires the production of exogenous antisense mRNA, generated either constitutively or inducibly, from a functional promoter. Classically, a constitutive promoter, such as the cytomegalovirus (CMV) promoter, is utilized to express high levels of antisense mRNA. This mRNA then binds irreversibly to endogenous sense mRNA, thus preventing its efficient translation, which results in attenuation of the protein product. Synthetic antisense oligo-nucleotides are also commonly used for constitutive antisense targeting. These DNA oligomers bind to endogenous RNA to generate RNA:DNA hybrids that are targeted by RNase H and degraded. This method will not be described at length in this chapter.

One problem often associated with constitutive antisense systems is low levels of expression, such that the system does not completely block translation of the targeted transcript. Another disadvantage is that the investigator has no temporal control over the antisense levels when using a constitutive expression system, because the promoter is always active. Therefore, the Tet-On and Tet-Off system is commonly utilized to circumvent these problems. This system allows researchers to exploit the tetracy-cline-regulated expressions systems originally described by Gossen and Bujard (TetOff) and Gossen et al. (Tet-On) and to regulate both spatially and temporally the levels of inductive antisense expression (1-6).

In order to obtain a Tet-Off or Tet-On cell line, the generation of a double stable cell line is required (3-6). First, the cell line is transfected with a regulatory plasmid. This vector expresses a transactivator element (tTA) for the Tet-On system, or a reverse transactivator element (rtTA), for the Tet-Off system, which is produced from the fusion of a tet repressor with the VP16 activation domain of the Herpes simplex virus,

From: Methods in Molecular Biology, Vol. 171:Proteoglycan Protocols Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

Tet Minimal Cmv Promoter

Fig. 1. Schematic representation of gene regulation in the Tet-Off and Tet-On Systems. (A) Under the control of the CMV promoter, the tet-tesponsive transcriptional activator (tTA), which is a fusion protein of the tetracycline repressor (TetR) and VP16 activation domain of the Herpes simplex virus, will bind to a tetracycline responsive element (TRE) in the absence of tetracycline or its derivative doxycycline (dox). The TRE contains seven copies of the 42-bp tet operator and is located upstream of the minimal CMV promoter (Pmin) which drives expression of a specific cDNA. (B) The reverse tet-responsive transcriptional activator (rtTA) differs from the tTA by 4 amino acid changes, which result in a reversal of its response to tetracyline and its derivates. Therefore, transcription is turned on in the presence of doxycyline through the binding of rtTA to the TRE. Reprinted with permission from Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W., and Bujard, H. (1995) Transcriptional activation by tetracyclines in mammalian cells Science 268, 1766-1769. Copyright ©1995 American Association for the Advancement of Science.

Fig. 1. Schematic representation of gene regulation in the Tet-Off and Tet-On Systems. (A) Under the control of the CMV promoter, the tet-tesponsive transcriptional activator (tTA), which is a fusion protein of the tetracycline repressor (TetR) and VP16 activation domain of the Herpes simplex virus, will bind to a tetracycline responsive element (TRE) in the absence of tetracycline or its derivative doxycycline (dox). The TRE contains seven copies of the 42-bp tet operator and is located upstream of the minimal CMV promoter (Pmin) which drives expression of a specific cDNA. (B) The reverse tet-responsive transcriptional activator (rtTA) differs from the tTA by 4 amino acid changes, which result in a reversal of its response to tetracyline and its derivates. Therefore, transcription is turned on in the presence of doxycyline through the binding of rtTA to the TRE. Reprinted with permission from Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W., and Bujard, H. (1995) Transcriptional activation by tetracyclines in mammalian cells Science 268, 1766-1769. Copyright ©1995 American Association for the Advancement of Science.

under the control of the CMV promoter (Fig. 1). When tetracycline, or its more commonly used derivative doxycycline, is added to the culture medium, the transactivator is induced to bind the tetracycline response element (TRE) located on the response plasmid (Fig. 1). This plasmid contains a multiple cloning site (MCS) located immediately downstream of the Tet-responsive minimal promoter. The promoter consists of the TRE, which contains seven copies of the 42-bp tet operator sequence (tetO) residing upstream of the minimal CMV promoter (-Pm;nCMV), which lacks the enhancer region of the full-length CMV promoter. Therefore, the TRE, in conjunction with the minimal CMV promoter, drives expression of a specific cDNA only upon binding of the tetR or rTetR to the tetO sequence (Fig. 2). Both Tet-On and Tet-Off utilize the

Tet Minimal Cmv Promoter

Fig. 2. Schematic representation of generation of double stable cell line. Cells are grown to confluence and transfected with a pTet-On or pTet-Off regulatory plasmid. Neomycin-resistant cells are cloned and expanded. Transient transfection with a luciferase construct (pTet-Luc) is performed to determine low-background and high-inducibility of the clones. The most optimal clone is then transfected with the response plasmid expressing specific antisense under the control of the TRE and the minimal CMV promoter. Hygromycin-resistant clones are then isolated and expanded.

Fig. 2. Schematic representation of generation of double stable cell line. Cells are grown to confluence and transfected with a pTet-On or pTet-Off regulatory plasmid. Neomycin-resistant cells are cloned and expanded. Transient transfection with a luciferase construct (pTet-Luc) is performed to determine low-background and high-inducibility of the clones. The most optimal clone is then transfected with the response plasmid expressing specific antisense under the control of the TRE and the minimal CMV promoter. Hygromycin-resistant clones are then isolated and expanded.

pTRE response plasmid. However, for the Tet-On system, the tTA activates transcription in the presence of doxycycline. Conversely, in the Tet-Off system, the rtTA, which has four amino acids mutated in the tet repressor, blocks transcription in the presence of doxycycline.

One advantage of the tetracycline inducible system is that it allows for the tight regulation of antisense expression with the presence of the TRE on the response plasmid. By incubating the clones with a minimal dose of doxycycline, it is possible to control when antisense expression is induced. Expression levels can then be assayed by Western blotting with an antibody specific to your protein, RT-PCR using gene-specific primers, Northern blot analysis, or a functional assay specific for your system. Once a stably transfected cell line has been generated, it is then possible to do more in-vitro and in-vivo analyses, including injection of inducible cells into mice followed by oral administration of doxycycline to induce expression (7-8). A second advantage of this system is that doxycycline is not toxic to the cells or to animals, and it is only slightly cytotoxic at relatively high levels (milligram amounts). Recent data also show evidence for the controlled expression of two genes in a mutually exclusive manner by utilizing this technology (9). Overall, this system has proven to be extremely beneficial in specific areas of research, and it ultimately has the potential to be utilized for therapeutic purposes.

2. Materials 2.1. General

1. DMEM (Life Technologies, Inc.).

2. Doxycycline (Sigma).

3. Fetal bovine serum (HyClone Laboratories, Inc.).

4. Glutamine (Mediatech, Inc.).

5. G418 (Geneticin, Sigma).

6. Hygromycin (Sigma).

7. Luciferase Assay Kit (Clontech).

8. Penicillin/streptomycin (Life Technologies, Inc.).

9. Serum-free defined media (Life Technologies, Inc.).

10. 50x TAE electrophoresis buffer, pH 8.5: 242 g Tris base, 57.1 mL glacial acetic acid, 37.2 g Na2EDTA-2H2O, made up to 1 L with distilled H2O.

11. TE buffer, pH 7.5: 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, pH 8.0.

12. Trypsin (Life Technologies, Inc.).

13. Tet-On/Tet-Off expression system (Clontech).

14. Tissue culture dishes (12 well, 6 well, 100 mm).

15. Loading buffer: 50% glycerol, 0.2 M EDTA, pH 8.3, 0.05% (w/v) bromophenol blue.

3. Methods

3.1. General

As mentioned earlier, the overall concept of this system is to generate a double stable cell line that will inducibly express specific antisense mRNA (see Fig. 2 for a general scheme). First, cells are stably transfected with a response plasmid expressing either the tTA or rtTA. Second, neomycin-resistant clones from the first transfection are then transiently transfected with a reporter plasmid, which is under the control of the TRE and the minimal CMV promoter, to measure the induciblity and background levels of the selected clones. In this case, we describe the utilization of the luciferase gene as a reporter and subsequent measurement of its activity with the luciferase assay. Third, after a suitable clone with low background and high inducibility has been selected, it is again stably transfected with the response plasmid, which contains a specific cDNA driven by the TRE and the minimal CMV promoter. Transcription is then regulated by the presence of tetracycline and its derivatives. These protocols will be described in more detail below.

3.2. Construction of Antisense Response Plasmid Vector

The most critical aspect of this procedure is determining the best region of the gene to target with the antisense mRNA. Often, several regions of the gene need to be tested before finding the most optimal. In general, investigators typically utilize the most conserved region in an effort to inhibit nonspecific hybridization. Cloning of cDNA can be accomplished by PCR amplification from a cDNA library, RT-PCR with gene-specific primers, or screening of a cDNA library.

1. In a sterile Eppendorf tube, digest 1-2 ^g of cDNA and response plasmid with compatible enzymes. If cDNA is cloned from a library, the sequence must be scanned for restriction sites that are located in the MCS of the response plasmid. There are several computer programs available, such as DNA Strider and PC Gene, which can be very helpful. If the cDNA is generated either by PCR from a cDNA library or RT-PCR from total RNA, it is possible to introduce novel restriction sites into the cloned sequences with the specifically designed primers.

2. Run digests on agarose gel. Agarose gel can be made by weighing out the necessary amount of agarose (typically a 1% gel will suffice) and add to TAE buffer. For these purposes, a small agarose gel can be utilized (~150 mL). Microwave until the agarose is dissolved, add ethidium bromide (0.5 ^g/mL), pour into gel tray and cool at room temperature until polymerized.

3. Add 2 ^L of 10x loading buffer (50% glycerol, 0.2 M EDTA, pH 8.3, 0.05% (w/v) bro-mophenol blue) to each sample and run on agarose gel along with the appropriate DNA ladder marker for 1.5-2 h at ~90 V.

4. When the gel is finished running, visualize the DNA by long-wave UV light and isolate the correct size cDNA fragment and linearized response plasmid with a clean razor blade. Purify the DNA and resuspend the DNA in an appropriate volume of TE.

5. Ligate the cDNA insert (0.5-1 ^g) into response plasmid (0.1 ^g) with T4 DNA ligase for 6 h at 13°C. Transform 1 ^L of ligation into Escherichia coli bacteria according to standard protocol and spread onto ampicillin-containing LB agar plates. Allow to grow at 37°C for at least 12 h.

6. Select 10-15 ampicillin colonies from the transformed plates and grow in 3 mLs of LB with ampicillin (50 mg/mL). Purify the plasmid by standard miniprep protocol. Correct orientation can be determined by appropriate restriction endonuclease digestion.

3.3. Generation of Stable Neomycin-Resistant Clones

The first stable transfection is with the regulator plasmid, containing either the tTA or the rtTA driven by the CMV promoter, as well as the neomycin resistance gene. Following transfection, clones are selected based on their ability to survive in G418.

1. One vial of cells is thawed and plated onto a 100-mm tissue culture dish containing suitable culture medium. Cells should be grown and/or passaged until they reach a density of approx 108 (see Note 1).

2. Cells are then trypsinized and then mixed with 40 ^g of the pTet-On regulatory plasmid DNA (Clontech) in a 0.4-mL cuvette. Cells are then transfected by electroporation in a BioRad Gene Pulsar according to the manufacturer's suggested conditions (see Note 2).

3. Following electroporation, cells are allowed to recover for ~10 min on ice and are then plated evenly on ten 100-mm tissue culture dishes. Use 10 mL of medium per dish (see Note 3).

4. Allow cells to attach and grow in a 37°C incubator (5% CO2).

5. After 48 h, G418 (400 ^g/mL) can be added to the culture medium. Media should be changed every 3-4 d (see Note 4 and Note 5).

6. After G418-resistant clones have reached a suitable size, 60-70 should be isolated by ring cloning. They can be plated directly into 12-well dishes. When confluent, cells can be expanded to 35-mm dishes (see Note 6).

3.4. Luciferase Assay

Following the first stable transfection, neomycin-resistant clones are transiently transfected with the plasmid pTRE-LUC. This plasmid is similar to the response plasmid except that the luciferase gene is expressed in place of a specific cDNA under the control of the TRE and the minimal CMV promoter. By measuring the level of luciferase activity with a functional assay, it is possible to determine which individual clones display the highest level of inducibility as well as the lowest background. This allows the investigator to screen the clones quickly before performing a second stable transfection.

1. Transiently transfect each neomycin resistant clone with 10 ^g of pTRE-LUC plasmid according to calcium phosphate protocol. We recommend using the CalPhos Maximizer (Clontech) for maximal transfection efficiency. Although we have suggested introducing the luciferase vector via calcium phosphate transfection, electroporation can also be utilized (see Note 5).

2. Cells are grown in 35-mm dishes ± doxycycline (2 ^g/mL) until 90% confluent.

3. Growth medium is removed from the cells and then rinsed twice with 1x PBS. Then 500 ^L of lysis buffer (25 mM Tris-phosphate, pH 7.8, 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N,N,N,N-tetracetic acid, 10% glycerol, 1% Triton X-100) are added to each dish and the cells are scraped off and placed in a sterile Eppendorf tube.

4. Tubes are then spun briefly (~5 s) in a Beckman microcentrifuge at 12,000g to pellet large debris.

5. Next, 20 ^L of room-temperature cell extract is mixed with 100 ^L of room-temperature Luciferase Assay Reagent (Promega).

6. The sample is placed in a luminometer to measure the release of light as the luciferin is being oxidized (see Note 7).

3.5. Generation of Antisense Inducible Clones

Once a clone has been selected to have low background levels and high inducibility based on the luciferase assay results, it undergoes a second stable transfection. It is transfected with two plasmids: a response plasmid containing specific cDNA under the control of the TRE and minimal CMV promoter, as well as a plasmid expressing the hygromycin resistance gene. Because the cells are already neomycin resistant, it is necessary to use a different antibiotic resistance marker to select clones.

1. The most optimal stably transfected clone, as determined by luciferase assay, is cotransfected with 40 ^g of the pTRE construct and 2 ^g of the hygromycin plasmid by electroporation as described previously. Because the clone is already G418 resistant, hygromycin is needed as a second antibiotic resistance marker.

2. After 48 h, 200 ^g/mL hygromycin was added to the culture medium (see Note 4).

3. Then 30-40 hygromycin-resistant clones are isolated by ring cloning and expanded as described previously.

3.6. Southern Blot Analysis

Although the clones have been selected through antibiotic resistance, it is also important to show that the regulatory and response plasmids have in fact integrated into the genome. This can be achieved by PCR with primers that amplify a specific region of the regulatory and response plasmids or by Southern analysis utilizing these plasmids as probes. We have found that Southern analysis is a much more reliable although labor-intensive method. It is preferable to perform Southern analysis after the first stable transfection as well before proceeding with the second stable transfection.

1. Extract genomic DNA from a 35-mm dish of each clone to be screened. DNA should be phenol:chloroformed and ethanol precipitated prior to restriction digestion.

2. Separate endonuclease digestions should be performed with an enzyme(s) that will liberate a distinct region within either the response plasmid and/or the regulatory plasmid. Digest 10 ^g of genomic DNA overnight at 37°C, followed by inactivation with 0.5 ^L of 0.5 M EDTA, phenol:chloroform extraction and ethanol precipitation.

3. Genomic digestions are run on a 0.8% agarose gel in TAE buffer at 80 V for 4-5 h. Gel percentage and duration of electrophoresis will depend on the size of the fragments that are expected.

4. Plasmid DNA should be digested with the same enzymes as genomic DNA and inserts purified as described previously.

5. After gel is finished running, it is denatured in 1.5 M NaCl and 0.5 M NaOH for 30 min at room temperature and then neutralized in 1 M ammonium acetate and 0.5 M NaOH for 30 min at room temperature. The DNA is transferred onto a nylon membrane in 10x SSC overnight and then UV cross-linked in a Stratalinker.

6. The plasmid probes should be labeled with 32P dCTP or, if preferred, a nonradioactive isotope such as digoxigenin, by random priming with Klenow according to the manufacturer's protocol.

7. Genomic DNA filters are prehybridized at 65°C for 3 h and then hybridized with the corresponding DNA probes at 65 °C overnight. Membranes are then washed and exposed to autoradiography film. Development of the film should yield the expected results.

3.7. Northern Blot Analysis

Northern analysis is needed to determine that the tTA or rtTA and the antisense cDNA are expressed. This will confirm expression of both the regulatory and response transcripts. Proper controls include extraction of total RNA in both the presence and absence of doxycycline. Once again, Northern analysis should ideally also be performed after the first stable transfection.

1. Total RNA is extracted from 35-mm confluent dishes of the clones to be screened by commonly used guanidium thiocyanate protocols (e.g., TRI-Reagent, Sigma) (see Note 8).

2. Then 20 ^g of total RNA are run on a 1% agarose/formaldehyde denaturing gel in MOPS buffer at 70 V for 4-5 h.

3. Total RNA is transferred onto a nylon filter and cross-linked with a UV Stratalinker. Filters are prehybridized at 42°C for 3 h and hybridized at 42°C overnight with the probes labeled the same as described for Southern analysis.

4. Membranes are then washed and exposed to autoradiography film. Development of the film should yield the expected results.

3.8. Western Immunoblotting Analyses

Western analysis is necessary to determine that there is a decrease in specific protein expression upon addition or removal of doxycycline. Which system is being used (Tet-On or Tet-Off) will dictate whether expression should be blocked with or without doxycycline. It is also possible to perform functional assays for the protein as well.

1. Antisense transfected clones are expanded into 35-mm dishes and then incubated for 48 h in serum-free defined media with or without 2 ^g/mL doxycycline.

2. Serial dilutions of the conditioned media from each clone are vacuum blotted onto a nylon filter (Schleicher and Scheull).

3. The filter is then blocked in 3% milk for 3 h and then incubated overnight at room temperature with a primary antibody to Protein X.

4. After several washes, the blot is incubated with an appropriate horseradish peroxidase-conju-gated secondary antibody.

5. Enhanced chemiluminescence is performed according to the manufacturer's protocol (Amersham).

6. Autoradiographs are then scanned by laser densitometry and quantitated to determine the relative protein levels.

4. Notes

1. When working with tissue cultures, sterility is of concern. Although antibiotics are added to the culture media, be sure to wipe down the hood with 70% ethanol before and after use, and wear gloves throughout the procedure.

2. Any DNA to be transfected into cells should be of highest quality. That is, after plasmid preparation, DNA should be phenol:chloroformed and ethanol precipitated under a sterile tissue culture hood. Also, as a final check, it is a good idea to run an aliquot on an agarose gel prior to transfection.

3. Be sure to determine the correct capacitance and voltage for the cell line and electroporator.

4. Due to variations in potency between different lots, it may be necessary to titrate the proper concentrations of G418 and hygromycin for each cell line.

5. It is important to mention that certain cell lines can alter the effectiveness of the tetracycline controlled gene expression (11). The type of transfection method as well as the amount of plasmid that is transfected can also alter considerably the background levels and inducibility of each clone. Therefore, it may be necessary to test several experimental parameters for each cell line and screen many clones in order to find one that is highly inducible but also gives very low levels of background expression. Although labor intensive, the results will be much more trustworthy.

6. When ring cloning, we have found that plastic rings work better than metal rings and require less vacuum grease to attach to the plate. Also, while picking clones, try to work rapidly, because the plates can dry out quickly and the cells will die.

7. We have noticed that often there are extremely high levels of background luciferase expression in many of the selected clones. Therefore, it may be necessary to select more than the recommended number of clones until one of low background can be isolated.

8. Be certain when working with RNA that gloves are always worn and are changed frequently. RNA is very susceptible to degradation from RNases that are found on most surfaces, including skin.

References

1. Gossen, M. and Bujard, H. (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. (USA) 89, 5547-5551.

2. Gossen, M., Bonin, A. L., and Bujard, H. (1993) Control of gene activity in higher eukary-otic cells by prokaryotic regulatory elements. TIBS 18, 471-475.

3. Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W., and Bujard, H. (1995) Transcriptional activation by tetracyclines in mammalian cells. Science 268, 1766-1769.

4. Baron, U., Freundlieb, S., Gossen, M., and Bujard, H. (1995) Co-regulation of two gene activities by tetracycline via a bidirectional promoter. Nucleic Acids Res. 23, 3605-3606.

5. Baron, U., Gossen, M., and Bujard, H. (1997) Tetracycline-controlled transcription in eukaryotes: novel transactivators with graded transactivation potential. Nucleic Acids Res. 25, 2723-2729.

6. Freundlieb, S., Baron, U., Bonin A.L., Gossen, M., and Bujard, H. (1997) Use of tetracy-cline-controlled gene expression systems to study mammalian cell cycle. Meth. Enzymol. 283, 159-173.

7. Furth, P. A., Onge, L. S., Boger, H., Gruss, P., Gossen, M., Kistner, A., Bujard, H., and Henninghausen, L. (1994) Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc. Natl. Acad. Sci. (USA) 91, 9302-9306.

8. Kistner, A., Gossen, M., Zimmermann, F., Jerecic, J., Ullmer, C., Lubbert, H., and Bujard, H. (1996) Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice. Proc. Natl. Acad. Sci. (USA) 93, 10,933-10,945.

9. Baron, U., Schnappinger, D., Helbl, V., Gossen, M., Hillen, W., and Bujard, H. (1999) Generation of conditional mutants in higher eukaryotes by switching between the expression of two genes. Proc. Natl. Acad. Sci. (USA) 96, 1013-1018.

10. Bonin, A. L., Gossen, M., and Bujard, H. (1994) Photinus pyralis luciferase: vectors that contain a modified luc coding sequence allowing convenient transfer into other systems. Gene 141, 75-77.

11. Gossen, M. and Bujard, H. (1995) Efficacy of tetracycline-controlled gene expression is influenced by cell type: commentary. BioTechniques 19, 213-216.

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