Inducible Protein Expression—T-REx System

Overview

The Invitrogen T-REx System is a tetracycline-regulated mammalian expression system that uses regulatory elements from the E. coli Tn10-encoded tetracycline (Tet) resistance operon (Hillen and Berens, 1994; Hillen et al., 1983). Tetracycline regulation in the T-REx System is based on the binding of tetracycline to the Tet repressor and derepression of the promoter controlling expression of the gene of interest (Yao et al., 1998). The major components of the T-REx System include:

• An inducible expression plasmid for expression of your gene of interest under the control of the strong human cytomegalovirus immediate-early (CMV) promoter and two tetracycline operator 2 (TetO2) sites
• A regulatory plasmid, pcDNA6/TR™, which encodes the Tet repressor (TetR) under the control of the human CMV promoter Tetracycline for inducing expression
• A control expression plasmid containing the lacZ gene, which when cotransfected with pcDNA6/TR™, expresses β-galactosidase upon induction with tetracycline.

For specific information on the inducible expression vector and the corresponding positive control vector containing the lacZ gene, please refer to the manual for the inducible expression vector you are using.

Description of the T-REx System

In the T-REx System, expression of your gene of interest is repressed in the absence of tetracycline and induced in the presence of tetracycline (Yao et al., 1998). Unlike other tetracycline-regulated systems which use hybrid regulatory molecules and viral transactivation domains (Gossen and Bujard, 1992), the T-REx System uses only regulatory elements from the native Tet operon (Yao et al., 1998). Tetracycline-regulated gene expression in the T-REx System more closely resembles the regulation of the native bacterial tet operon (Hillen and Berens, 1994; Hillen et al., 1983) and avoids the potentially toxic effects of viral transactivation domains observed in some mammalian cell lines.

The major component of the T-REx System is the inducible expression plasmid. Expression of your gene of interest from the inducible expression vector is controlled by the strong CMV promoter (Andersson et al., 1989; Boshart et al., 1985; Nelson et al., 1987) into which 2 copies of the tet operator 2 (TetO2) sequence have been inserted in tandem. The TetO2 sequences consist of 2 copies of the 19 nucleotide sequence, 5´-TCCCTATCAGTGATAGAGA-3´ separated by a 2 base pair spacer (Hillen and Berens, 1994; Hillen et al., 1983). Each 19 nucleotide TetO2 sequence serves as the binding site for 2 molecules of the Tet repressor. For more information about the Tet operator sequences and the specific features of each inducible expression vector, please refer to the manual for the vector you are using.

The second major component of the T-REx System is the pcDNA6/TR regulatory vector which expresses high levels of the TetR gene (Postle et al., 1984) under the control of the human CMV promoter. Both T-REx vectors can be introduced into mammalian host cells by standard transfection methods.

Mechanism of repression

In the absence of tetracycline, the Tet repressor forms a homodimer that binds with extremely high affinity to each TetO2 sequence in the promoter of the inducible expression vector (Hillen and Berens, 1994). The 2 TetO2 sites in the promoter of the inducible expression vector serve as binding sites for 4 molecules (or 2 homodimers) of the Tet repressor. The affinity of the Tet repressor for the tet operator is KB = 2 x 1011 M-1 (as measured under physiological conditions), where KB is the binding constant (Hillen and Berens, 1994). Binding of the Tet repressor homodimers to the TetO2 sequences represses transcription of your gene of interest. Upon addition, tetracycline binds with high affinity to each Tet repressor homodimer in a 1:1 stoichiometry and causes a conformational change in the repressor that renders it unable to bind to the Tet operator. The association constant, KA, of tetracycline for the Tet repressor is 3 x 10⁹ M-1 (Hillen and Berens, 1994). The Tet repressor: tetracycline complex then dissociates from the Tet operator and allows induction of transcription from the gene of interest.

Experimental outline

The gene of interest is cloned into the multiple cloning site of the inducible expression vector, and the resulting construct cotransfected with the regulatory plasmid, pcDNA6/TR™ into mammalian cells. After transfection, cells are treated with tetracycline to derepress the hybrid CMV/TetO2 promoter in the inducible expression vector and induce transcription of your gene of interest.

The positive control vector containing the lacZ gene can be transiently cotransfected into mammalian cells with pcDNA6/TR™ to demonstrate that the system is working properly in your cell line. Stable cell lines expressing Tet repressor from pcDNA6/TR™ can be established to serve as hosts for inducible expression vector-based constructs.

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The T-REx System manual is supplied with the kits listed below. The Core System includes the inducible expression vector of choice, the regulatory vector, and primers for sequencing. The Complete System includes the Core System plus inducing and selection agents. See below for a detailed description of the contents of each T-REx Kit.

Shipping/storage

The T-REx Core System is shipped at room temperature. Store at -20°C.

The T-REx Complete System is shipped in 2 boxes. Store as described below:

• Box 1 contains vectors, primers, and blasticidin and is shipped at room temperature. Upon receipt, remove the vectors and primers and store at -20°C. Blasticidin powder should be stored at +4°C.
• Box 2 contains tetracycline and Zeocin and is shipped on blue ice. Store at +4°C. For long-term storage (> 6 months), store at -20°C. Store the tetracycline and Zeocin protected from exposure to light.

Kit contents

Both the T-REx Complete and the T-REx Core Systems include the following regulatory vector and sequencing primers. Store at -20°C.

Inducible expression vector

Each T-REx Complete and Core System also includes one of the following inducible expression vectors and a corresponding positive control vector containing the lacZ gene. Please refer to the vector manual for specific information pertaining to each inducible expression vector. Store at -20°C.

T-REx cell lines

For your convenience, Invitrogen has available three mammalian cells lines that stably express the Tet repressor. T-REx-293 cells and T-REx-HeLa cells express the Tet repressor from pcDNA6/TR™ and should be maintained in medium containing blasticidin. T-REx-U2OS cells express the Tet repressor from pCEP4/tetR as described in Yao et al., 1998, and should be maintained in medium containing hygromycin. Please note that the pCEP4/tetR plasmid in the T-REx-U2OS cells is episomally-maintained, but is stable under hygromycin selection. For more information, please go to www.thermofisher.com or call Technical Service.

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Introduction

The following section contains guidelines for maintaining and propagating the pcDNA6/TR regulatory vector.

General molecular biology techniques

For assistance with E. coli transformations, restriction enzyme analysis, purification of single-stranded DNA, DNA sequencing, and DNA biochemistry, please refer to Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989) or Current Protocols in Molecular Biology (Ausubel et al., 1994).

E. coli strain

Many E. coli strains are suitable for the propagation of the pcDNA6/TR vector including TOP10F´ (Catalog no. C615-00), DH5αF´, and INVαF´ (Catalog no. C658?00). We recommend that you propagate the pcDNA6/TR vector in E. coli strains that are recombination deficient (recA) and endonuclease A deficient (endA).

For your convenience, TOP10F´ E. coli are available as chemically competent or electrocompetent cells from Invitrogen.

Transformation method

You may use any method of choice for transformation. Chemical transformation is the most convenient for many researchers. Electroporation is the most efficient and the method of choice for large plasmids.

Maintenance of plasmids

The pcDNA6/TR™ vector contains the ampicillin and blasticidin resistance genes to allow selection of the plasmid using ampicillin or blasticidin. To propagate and maintain the pcDNA6/TR plasmid, we recommend using the following procedure:

1. Resuspend the vector in 20 µL sterile water to prepare a 1 µg/µL stock solution. Store the stock solution at -20°C.
2. Use the stock solution to transform a recA, endA E. coli strain like TOP10F´, INVαF´, or equivalent.
3. Select transformants on LB agar plates containing 50 to 100 µg/mL ampicillin or 50 µg/mL blasticidin in Low Salt LB.
4. Prepare a glycerol stock of each plasmid for long-term storage (see protocol below).

Selection in E. coli

To facilitate selection of blasticidin-resistant E. coli, the salt concentration of the medium must remain low (< 90 mM) and the pH must be 7.0. Prepare Low Salt LB broth and plates using the recipe. Failure to lower the salt content of your LB medium will result in non-selection due to inhibition of the drug unless a higher concentration of blasticidin is used.

Preparing a glycerol stock

Once you have identified the correct clone, be sure to purify the colony and make a glycerol stock for long-term storage. It is also a good idea to keep a DNA stock of your plasmid at -20°C.

1. Streak the original colony out on an LB plate containing 50 µg/mL ampicillin or 100 µg/mL blasticidin in Low Salt LB. Incubate the plate at 37°C overnight.
2. Isolate a single colony and inoculate into 1–2 mL of LB containing 50 µg/mL ampicillin or 50 µg/mL blasticidin in Low Salt LB.
3. Grow the culture to mid-log phase (OD600 = 0.5–0.7).
4. Mix 0.85 mL of culture with 0.15 mL of sterile glycerol and transfer to a cryovial.
5. Store at -80°C.

Plasmid preparation

Plasmid DNA for transfection into eukaryotic cells must be very clean and free from phenol and sodium chloride. Contaminants will kill the cells, and salt will interfere with lipids decreasing transfection efficiency. We recommend isolating DNA using the S.N.A.P. MiniPrep Kit (10–15 µg DNA, Catalog no. K1900-01), the S.N.A.P. MidiPrep Kit (10–200 µg DNA, Catalog no. K1910-01) or CsCl gradient centrifugation.

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Introduction

The following section contains guidelines for maintaining and propagating the pcDNA6/TR regulatory vector.

General molecular biology techniques

For assistance with E. coli transformations, restriction enzyme analysis, purification of single-stranded DNA, DNA sequencing, and DNA biochemistry, please refer to Molecular Cloning: A Laboratory Manual (Sambrook et al., 1989) or Current Protocols in Molecular Biology (Ausubel et al., 1994).

E. coli strain

Many E. coli strains are suitable for the propagation of the pcDNA6/TR vector including TOP10F´ (Catalog no. C615-00), DH5αF´, and INVαF´ (Catalog no. C658?00). We recommend that you propagate the pcDNA6/TR vector in E. coli strains that are recombination deficient (recA) and endonuclease A deficient (endA).

For your convenience, TOP10F´ E. coli are available as chemically competent or electrocompetent cells from Invitrogen.

Transformation method

You may use any method of choice for transformation. Chemical transformation is the most convenient for many researchers. Electroporation is the most efficient and the method of choice for large plasmids.

Maintenance of plasmids

The pcDNA6/TR™ vector contains the ampicillin and blasticidin resistance genes to allow selection of the plasmid using ampicillin or blasticidin. To propagate and maintain the pcDNA6/TR plasmid, we recommend using the following procedure:

1. Resuspend the vector in 20 µL sterile water to prepare a 1 µg/µL stock solution. Store the stock solution at -20°C.
2. Use the stock solution to transform a recA, endA E. coli strain like TOP10F´, INVαF´, or equivalent.
3. Select transformants on LB agar plates containing 50 to 100 µg/mL ampicillin or 50 µg/mL blasticidin in Low Salt LB.
4. Prepare a glycerol stock of each plasmid for long-term storage (see protocol below).

Selection in E. coli

To facilitate selection of blasticidin-resistant E. coli, the salt concentration of the medium must remain low (< 90 mM) and the pH must be 7.0. Prepare Low Salt LB broth and plates using the recipe. Failure to lower the salt content of your LB medium will result in non-selection due to inhibition of the drug unless a higher concentration of blasticidin is used.

Preparing a glycerol stock

Once you have identified the correct clone, be sure to purify the colony and make a glycerol stock for long-term storage. It is also a good idea to keep a DNA stock of your plasmid at -20°C.

1. Streak the original colony out on an LB plate containing 50 µg/mL ampicillin or 100 µg/mL blasticidin in Low Salt LB. Incubate the plate at 37°C overnight.
2. Isolate a single colony and inoculate into 1–2 mL of LB containing 50 µg/mL ampicillin or 50 µg/mL blasticidin in Low Salt LB.
3. Grow the culture to mid-log phase (OD600 = 0.5–0.7).
4. Mix 0.85 mL of culture with 0.15 mL of sterile glycerol and transfer to a cryovial.
5. Store at -80°C.

Plasmid preparation

Plasmid DNA for transfection into eukaryotic cells must be very clean and free from phenol and sodium chloride. Contaminants will kill the cells, and salt will interfere with lipids decreasing transfection efficiency. We recommend isolating DNA using the S.N.A.P. MiniPrep Kit (10–15 µg DNA, Catalog no. K1900-01), the S.N.A.P. MidiPrep Kit (10–200 µg DNA, Catalog no. K1910-01) or CsCl gradient centrifugation.

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Introduction

Once you have established that your construct can be inducibly expressed, you may wish to establish a stable cell line that constitutively expresses the Tet repressor and inducibly expresses your gene of interest. We recommend that you first create a stable cell line that expresses only the Tet repressor, then use that cell line to create a second cell line that will express your gene of interest from the inducible expression plasmid. Alternatively, you can transfect with both plasmids (pcDNA6/TR and inducible expression vector) and dual-select with blasticidin and Zeocin to isolate a single stable cell line expressing both the Tet repressor and your gene of interest.

Three T-REx cell lines that stably express the Tet repressor are available from Invitrogen. If you wish to assay for tetracycline-inducible expression of your gene of interest in 293, HeLa, or U2OS cells, you may want to use one of the T-REx cell lines as the host to establish your double stable cell line. For more information, go to www.invitrogen.com or call Technical Service.

Reminder: When generating a stable cell line expressing the Tet repressor, you will want to select for clones that express the highest levels of Tet repressor to use as hosts for your inducible expression construct. Those clones that express the highest levels of Tet repressor should exhibit the most complete repression of basal transcription of your gene of interest.

Determination of antibiotic sensitivity

To successfully generate a stable cell line expressing the Tet repressor and your protein of interest, you need to determine the minimum concentration of each antibiotic (blasticidin and Zeocin) required to kill your untransfected host cell line. For each antibiotic, test a range of concentrations (see below) to ensure that you determine the minimum concentration necessary for your cell line. Use the protocol below to determine the minimal concentrations of Zeocin and blasticidin required to prevent growth of the parental cell line. Please refer to the Appendix, page 14 for instructions on how to prepare and store blasticidin. Instructions on how to prepare and store Zeocin can be found in the inducible expression vector manual.

1. Plate or split a confluent plate so the cells will be approximately 25% confluent. For each antibiotic, prepare a set of 6-7 plates. Add the following concentrations of antibiotic to each plate in a set:
   • For blasticidin selection, test 0, 1, 3, 5, 7.5, and 10 µg/mL blasticidin
   • For Zeocin selection, test 0, 50, 125, 250, 500, 750, and 1000 µg/mL Zeocin
2. Replenish the selective media every 3-4 days, and observe the percentage of surviving cells.
3. Count the number of viable cells at regular intervals to determine the appropriate concentration of antibiotic that prevents growth within 1-2 weeks after addition of the antibiotic.

Effect of Zeocin on sensitive and resistant cells

Zeocin's method of killing is quite different from other antibiotics including blasticidin, neomycin, and hygromycin. Cells do not round up and detach from the plate. Sensitive cells may exhibit the following morphological changes upon exposure to Zeocin:

• Vast increase in size (similar to the effects of cytomegalovirus infecting permissive cells)
• Abnormal cell shape
• Presence of large empty vesicles in the cytoplasm (breakdown of the endoplasmic reticulum and golgi apparatus, or other scaffolding proteins)
• Breakdown of plasma and nuclear membrane (appearance of many holes in these membranes)
• Eventually, these "cells" will completely break down and only "strings" of protein remain.
• Zeocin-resistant cells should continue to divide at regular intervals to form distinct colonies. There should not be any distinct morphological changes in Zeocin-resistant cells when compared to cells not under selection with Zeocin. For more information about Zeocin and its mechanism of action, please refer to the inducible expression vector manual.

Possible sites for linearization of pcDNA6/TR

To obtain stable transfectants, you may choose to linearize pcDNA6/TR before transfection. While linearizing the vector may not improve the efficiency of transfection, it increases the chances that the vector does not integrate in a way that disrupts the Tet repressor gene or other elements necessary for expression in mammalian cells. The table below lists unique sites that may be used to linearize your construct prior to transfection. Other restriction sites are possible.

Selection of stable integrants

Once you have determined the appropriate antibiotic concentrations to use for selection, you can generate a stable cell line expressing pcDNA6/TR and your inducible expression construct.

1. Transfect mammalian cells with pcDNA6/TR using the desired protocol. Remember to include a plate of untransfected cells as a negative control.
2. 24 hours after transfection, wash the cells and add fresh medium to the cells.
3. 48 hours after transfection, split the cells into fresh medium containing blasticidin at the pre-determined concentration required for your cell line. Split the cells such that the cells are no more than 25% confluent. If the cells are too dense, the antibiotic will not kill the cells. Antibiotics work best on actively dividing cells.
4. Feed the cells with selective medium every 3-4 days until foci can be identified.
5. Pick at least 20 foci and expand them to test for tetracycline-inducible gene expression by transiently transfecting with the positive control plasmid expressing beta-galactosidase. Screen for those clones that exhibit the lowest levels of basal transcription and the highest levels of β-galactosidase expression after addition of tetracycline.
6. Once you have obtained a stable cell line expressing the Tet repressor, you can use this cell line to isolate a stable cell line expressing your gene of interest from the inducible expression plasmid. Repeat Steps 1 through 4, above, using your inducible expression vector construct and Zeocin to select foci. Remember to maintain your cells in medium containing blasticidin as well.
7. Pick and expand at least 20 foci to test for tetracycline-regulated gene expression.

Dual selection of stable integrants

We recommend using the protocol above to generate double stable cell lines. However, if you wish to perform dual selection, you may cotransfect both pcDNA6/TR and your inducible expression construct into the cell line of choice. When cotransfecting your cell line, we recommend using a ratio of at least 6:1 (w/w) pcDNA6/TR DNA:inducible expression plasmid DNA to increase the chances that your double stable cell lines will express sufficient levels of Tet repressor. Following the steps listed above, add selective medium containing blasticidin and Zeocin at the concentrations determined previously. Screen at least 40 foci for tetracycline-regulated expression of your gene of interest using an appropriate assay for your protein (e.g. western blot, enzymatic assay).

Once you have isolated stable cell lines expressing both pcDNA6/TR™ and your inducible expression construct and have tested for tetracycline-inducible expression of your gene of interest, we recommend that you perform a time course of tetracycline induction to optimize expression of your protein of interest (e.g. 0, 8, 16, 24, 32 hours, etc.). Use the appropriate concentration of tetracycline for your cell line as previously determined. We have observed as much as 120-fold induction of β-galactosidase in a double stable cell line (T-REx 293 cells transfected with the positive control plasmid containing the lacZ gene) after 24 hours of treatment with 1 µg/mL tetracycline. Please note that your induction levels may vary depending on the nature of your gene of interest and the particular clone that you choose.

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Map of pcDNA6/TR

pcDNA6/TR is a 6662 bp vector that expresses the Tet repressor under the control of the human CMV promoter. A figure summarizing the features of this vector can be downloaded by clicking here. The complete sequence for pcDNA6/TR is available for downloading from www.thermofisher.com or by contacting Technical Service.

TetR gene

The TetR gene used in pcDNA6/TR was originally isolated from the Tn10 transposon which confers resistance to tetracycline in E. coli and other enteric bacteria (Postle et al., 1984). The TetR gene from Tn10 encodes a class B Tet repressor and is often referred to as TetR(B) in the literature (Hillen and Berens, 1994).

The TetR gene encodes a repressor protein of 207 amino acids with a calculated molecular weight of 23 kDa. For more information about the Tet repressor and its interaction with the Tet operator, please refer to the review by Hillen and Berens (1994).

Features of pcDNA6/TR

The table below describes the relevant features of pcDNA6/TR. The vector includes the rabbit β-globin intron II to enhance expression of the TetR gene. For a more detailed description of the TetR gene and the Tet repressor, please refer to the previous page. All features have been functionally tested.

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Blasticidin S HCl is a nucleoside antibiotic isolated from Streptomyces griseochromogenes which inhibits protein synthesis in both prokaryotic and eukaryotic cells (Takeuchi et al., 1958; Yamaguchi et al., 1965). Resistance is conferred by expression of either one of two blasticidin S deaminase genes: bsd from Aspergillus terreus (Kimura et al., 1994) or bsr from Bacillus cereus (Izumi et al., 1991). These deaminases convert blasticidin S to a non-toxic deaminohydroxy derivative (Izumi et al., 1991).

Molecular weight, formula, and structure

The formula for blasticidin S is C17H26N8O5-HCl, and the molecular weight is 458.9. The diagram below shows the structure of blasticidin.

Handling Blasticidin

Always wear gloves, mask, goggles, and protective clothing (e.g. a laboratory coat) when handling blasticidin. Weigh out blasticidin and prepare solutions in a hood.

Preparing and storing stock solutions

Blasticidin is supplied in the T-REx Complete Kit, but may also be obtained separately from Invitrogen (Catalog no. R210-01) in 50 mg aliquots. Blasticidin is soluble in water. Sterile water is generally used to prepare stock solutions of 5 to 10 mg/mL.

• Dissolve blasticidin in sterile water and filter-sterilize the solution.
• Aliquot in small volumes suitable for one time use (see next to last point below) and freeze at -20°C for long-term storage or store at +4°C for short-term storage.
• Aqueous stock solutions are stable for 1-2 weeks at +4°C and 6-8 weeks at -20°C.
• pH of the aqueous solution should be 7.5 to prevent inactivation of blasticidin.
• Do not subject stock solutions to freeze/thaw cycles (do not store in a frost-free freezer).
• Upon thawing, use what you need and store the thawed stock solution at +4°C for up to 2 weeks.
• Containing blasticidin may be stored at +4°C for up to 2 weeks.

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