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Clontech Laboratories, Inc.

Tet-On 3G Inducible Expression Systems User Manual

?

PT5148-1 (082012) Cat. Nos. Many

Clontech Laboratories, Inc. A Takara Bio Company 1290 Terra Bella Avenue, Mountain View, CA 94043, USA U.S. Technical Support: tech@clontech.com United States/Canada 800.662.2566 Asia Pacific +1.650.919.7300 Europe +33.(0)1.3904.6880 Japan +81.(0)77.543.6116

Tet-On? 3G Inducible Expression Systems User Manual

Table of Contents
I. II. Introduction..................................................................................................................................................................... 3 List of Components......................................................................................................................................................... 4

III. Additional Materials Required........................................................................................................................................ 5 IV. Protocol Overview .......................................................................................................................................................... 7 V. Cloning Your Gene of Interest into a pTRE3G Vector using In-Fusion HD.................................................................. 9 A. Materials Required ................................................................................................................................................. 10 B. Protocol .................................................................................................................................................................. 10 VII. Creating a Tet-On 3G-Expressing Stable Cell Line ..................................................................................................... 11 A. Materials Required ................................................................................................................................................. 11 B. Protocol: Transfect and Select for 24 Independent Clones .................................................................................... 11 C. Protocol: Testing Your Tet-On 3G Clones for Induction ....................................................................................... 12 VIII. Creating & Screening for a Double-Stable Cell Line Capable of High Induction of your GOI ................................... 13 A. Materials Required ................................................................................................................................................. 13 B. Protocol: Creating a Double-Stable Tet-On 3G Inducible Cell Line ..................................................................... 13 C. Protocol: Screening Your Double-Stable Tet-On 3G Inducible Cell Lines ........................................................... 14 IX. References..................................................................................................................................................................... 15 X. Troubleshooting ............................................................................................................................................................ 16 Appendix A: Tet-On 3G Systems Vector Information ......................................................................................................... 18 Appendix B: Why Use a Linear Selection Marker?.............................................................................................................. 21 Appendix C: Selecting Stable Clones via Limited Dilution of Suspension Cells ................................................................. 22 Appendix D: Preparing and Handling Tet-On 3G Cell Line Stocks ..................................................................................... 23 VI. Pilot Testing Tet-Based Induction of Your Construct .................................................................................................... 9

Table of Figures
Figure 1. The Tet-On 3G Systems allow inducible gene expression in the presence of Dox. ................................................ 3 Figure 2. Establishing the Tet-On 3G System in target cells. ................................................................................................. 8 Figure 3. The In-Fusion HD Single-Tube Cloning Protocol................................................................................................... 9 Figure 4. Transfection of the regulator and response plasmids into target cells in a 6-well plate. ....................................... 10 Figure 5. pCMV-Tet3G Vector and pEF1α-Tet3G Vector Maps. ........................................................................................ 18 Figure 6. pTRE3G Vector and pTRE3G-IRES Vector Maps. .............................................................................................. 18 Figure 7. pTRE3G-mCherry Vector and pTRE3G-ZsGreen1 Vector Maps. ....................................................................... 19 Figure 8. pTRE3G-Luc Control Vector Map. ....................................................................................................................... 19 Figure 9. pTRE3G-BI-Luc Control Vector and pTRE3G-BI Vector Maps. ......................................................................... 20 Figure 10. pTRE3G-BI-mCherry Vector and pTRE3G-BI-ZsGreen1 Vector Maps. ........................................................... 20

Table of Tables
Table 1. Recommended Antibiotic Concentrations for Selecting & Maintaining Stable Cell Lines ...................................... 5

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Tet-On? 3G Inducible Expression Systems User Manual

I.

Introduction
A. Summary
The Tet-On 3G Systems are inducible gene expression systems for mammalian cells. Target cells that express the Tet-On 3G transactivator protein and contain a gene of interest (GOI) under the control of a TRE3G promoter (PTRE3G) will express high levels of your GOI, but only when cultured in the presence of doxycycline (Dox) (Figure 1).

Figure 1. The Tet-On 3G Systems allow inducible gene expression in the presence of Dox.

B.

Two Elements of Tet-On 3G
Tet-On 3G Transactivator Protein Based on the transcriptional regulators described by Gossen & Bujard (1992), Gossen et al. (1995), and Urlinger et al. (2000), Tet-On 3G is a modified form of the Tet-On Advanced transactivator protein which has been evolved to display far higher sensitivity to doxycycline (Zhou et. al,. 2006). PTRE3G Inducible Promoter The inducible promoter PTRE3G provides for very low basal expression and high maximal expression after induction (L?w et. al., submitted). It consists of 7 repeats of a 19 bp tet operator sequence located upstream of a minimal CMV promoter. In the presence of Dox, Tet-On 3G binds specifically to PTRE3G and activates transcription of the downstream GOI. PTRE3G lacks binding sites for endogenous mammalian transcription factors, so it is virtually silent in the absence of induction.

C.

Doxycycline
Doxycycline is a synthetic tetracycline derivative that is the effector molecule for the Tet-On and Tet-Off? Systems. When bound by Dox, the Tet-On 3G protein undergoes a conformational change that allows it to bind to tet operator sequences located in the PTRE3G promoter (Figure 1). The Dox concentrations required for induction of Tet-On Systems are far below cytotoxic levels for either cell culture or transgenic studies, and Tet-On 3G responds to even lower concentrations than its predecessors (Zhou et. al,. 2006). Note that Tet-On Systems respond well only to doxycycline, and not to tetracycline (Gossen & Bujard, 1995). The half-life of Dox in cell culture medium is 24 hours. To maintain continuous inducible GOI expression in cell culture, the medium should be replenished with Dox every 48 hours.

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Tet-On? 3G Inducible Expression Systems User Manual

II.

List of Components
A. Available Tet-On 3G Plasmid Systems
Cat. No. 631168 631167 631166 631165 631164 631337 631338 631339 631340 631341 631342 631346 631347 631348 System Name Tet-On 3G Inducible Expression System Tet-On 3G Inducible Expression System (EF1α Version) Tet-On 3G Inducible Expression System (Bicistronic Version) Tet-On 3G Inducible Expression System (with mCherry) Tet-On 3G Inducible Expression System (with ZsGreen1) Tet-On 3G Bidirectional Inducible Expression System Tet-On 3G Bidirectional Inducible Expression System (with mCherry) Tet-On 3G Bidirectional Inducible Expression System (with ZsGreen1) Tet-On 3G Bidirectional Inducible Expression System (EF1alpha Version) Tet-On 3G Bidirectional Inducible Expression System (EF1alpha, mCherry) Tet-On 3G Bidirectional Inducible Expression System (EF1alpha, ZsGreen1) Tet-On 3G Inducible Expression System (EF1alpha, Bicistronic) Tet-On 3G Inducible Expression System (EF1alpha, mCherry) Tet-On 3G Inducible Expression System (EF1alpha, ZsGreen1)

B.

General System Components
All systems listed in Section II.A contain the following 7 components (store all components at -20°C): ? ? ? ? ? ? ? 10 ?g 10 ?g 10 ?g 2 ?g 2 ?g 100 rxns 50 ml regulator plasmid (see Section II.C) response plasmid (see Section II.C) control response plasmid pTRE3G-Luc or pTRE3G-BI-Luc (bidirectional systems) Linear Hygromycin Marker (also sold separately as Cat. No. 631625) Linear Puromycin Marker (also sold separately as Cat. No. 631626) Xfect? Transfection Reagent (also sold separately as Cat. No. 631317) Tet System Approved FBS, US Sourced (also sold separately as Cat. No. 631105)

C.

System-Specific Regulator and Response Plasmids
Cat. No. 631168 631167 631166 631165 631164 631337 631338 631339 631340 631341 631342 631346 631347 631348 Regulator Plasmid pCMV-Tet3G pEF1a-Tet3G pCMV-Tet3G pCMV-Tet3G pCMV-Tet3G pCMV-Tet3G pCMV-Tet3G pCMV-Tet3G pEF1a-Tet3G pEF1a-Tet3G pEF1a-Tet3G pEF1a-Tet3G pEF1a-Tet3G pEF1a-Tet3G Response Plasmid pTRE3G pTRE3G pTRE3G-IRES pTRE3G-mCherry pTRE3G-ZsGreen1 pTRE3G-BI pTRE3G-BI-mCherry pTRE3G-BI-ZsGreen1 pTRE3G-BI pTRE3G-BI-mCherry pTRE3G-BI-ZsGreen1 pTRE3G-IRES pTRE3G-mCherry pTRE3G-ZsGreen1

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Tet-On? 3G Inducible Expression Systems User Manual

III.

Additional Materials Required
A. Tetracycline-Free Fetal Bovine Serum
Contaminating tetracyclines, often found in serum, will significantly elevate basal expression when using Tet-On 3G. The following functionally tested tetracycline-free sera are available from Clontech:
Cat. No. 631106 631107 631101 631105 631040 631039 631158 631157 Serum Name Tet System Approved FBS (500 ml) Tet System Approved FBS (50 ml) Tet System Approved FBS, US-Sourced (500 ml) Tet System Approved FBS, US-Sourced (50 ml) Tet System Approved FBS, Australia-Sourced (500 ml) Tet System Approved FBS, Australia-Sourced (50 ml) Tet System Approved FBS, ES Cell Qualified (500 ml) Tet System Approved FBS, ES Cell Qualified (50 ml)

B.

Antibiotics for Selecting Stable Cell Lines
Table 1. Recommended Antibiotic Concentrations for Selecting & Maintaining Stable Cell Lines

Recommended Concentration (?g/ml) Cat. No. 631308 631307 631306 631305 631309
1

Antibiotic G418 (5 g) G418 (1 g) Puromycin (100 mg) Puromycin (25 mg) Hygromycin B (1 g)

Selecting Colonies 100–800

1

Maintenance 200

0.25–10 50–400

0.25 100

When selecting for single colonies, the appropriate dose must be determined empirically for your specific cell line. Test a dosage range using dishes of untransfected cells and choose the dose that kills all of the cells in 3–5 days. If all the cells die in less than 24 hr, you should use a lower dose.

C.

Tet-On 3G Cell Lines
Cat. No. 631183 631182 631181 631195 631197 Cell Line HeLa Tet-On 3G Cell Line HEK 293 Tet-On 3G Cell Line Jurkat Tet-On 3G Cell Line CHO Tet-On 3G Cell Line NIH/3T3 Tet-On 3G Cell Line

D.

Mammalian Cell Culture Supplies
? ? ? ? ? Culture medium, supplies, and additives specific for your target cells Trypsin/EDTA (e.g., Sigma, Cat. No. T4049) Cloning cylinders or discs for isolating colonies of adherent cell lines (Sigma, Cat. No. C1059) Cell Freezing Medium, with or without DMSO (Sigma, Cat. Nos. C6164 or C6039), for freezing Tet-On 3G cell lines. 6-well, 12-well & 24-well cell culture plates, 10 cm cell culture dishes
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Tet-On? 3G Inducible Expression Systems User Manual E. Doxycycline
? 5 g Doxycycline (Cat. No. 631311) Dilute to 1 mg/ml in double distilled H2O. Filter sterilize, aliquot, and store at –20°C in the dark. Use within one year.

F.

Xfect Transfection Reagents
Xfect Transfection Reagent provides high transfection efficiency and low cytotoxicity for most commonly used cell types. Xfect mESC Transfection Reagent is optimized for mouse embryonic stem cells.
Cat. No. 631317 631318 631320 631321 Transfection Reagent Xfect Transfection Reagent (100 rxns) Xfect Transfection Reagent (300 rxns) Xfect mESC Transfection Reagent (100 rxns) Xfect mESC Transfection Reagent (300 rxns)

G.

In-Fusion? HD Cloning System
In-Fusion is a revolutionary technology that greatly simplifies cloning. For more information, visit www.clontech.com/infusion
Cat. No. 639645 639646 639647 In-Fusion Cloning Kit In-Fusion HD Cloning System (10 rxns) In-Fusion HD Cloning System (50 rxns) In-Fusion HD Cloning System (100 rxns)

H.

Stellar? Competent Cells
We recommend using Stellar Competent Cells (see Section V), which are included in the In-Fusion HD Cloning Kits listed in Section III.G. You can also purchase these cells separately (Cat. No. 636763). The Cla I restriction site in the pTRE3G and pTRE3G-IRES vectors is blocked by an overlapping dam methylation site. Therefore, in order to digest these vectors with Cla I, you must first propagate them in a dam– bacterial strain such as Clontech’s (dam–/dcm–) Stellar Competent Cells (Cat. No. 636764), which must be purchased separately.

I.

TetR Monoclonal Antibody
If you wish to confirm that Tet-On 3G is expressed in your cells, we recommend that you use the following antibody and detect the protein via Western Blot.
Cat. No. 631131 631132 Antibody TetR Monoclonal Antibody (Clone 9G9) (40 ?g) TetR Monoclonal Antibody (Clone 9G9) (200 ?g)

J.

Luciferase Assay and Luminometer
These items are required when using the pTRE3G-Luc Vector to screen Tet-On 3G clones (Section VII.C). Use any standard luciferase assay system and luminometer.

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Tet-On? 3G Inducible Expression Systems User Manual

IV.

Protocol Overview
Please read each protocol completely before starting. Successful results depend on understanding and performing the following steps correctly.

A.

General Cell Culture
1. This user manual provides only general guidelines for mammalian cell culture techniques. For users requiring more information on mammalian cell culture, transfection, and creating stable cell lines, we recommend the following general reference: Freshney, R.I. (2005). Culture of Animal Cells: A Manual of Basic Technique, 5th Edition (WileyLiss, Hoboken, NJ). 2. The premade Tet-On 3G-expressing cell lines (Section III.C) save time and provide high performance when creating an inducible system. They have been prescreened and selected for high inducibility. NOTE: Skip Section VII if you have purchased one of these cell lines. Instead, see the Tet Cell Lines Protocol-at-a-Glance (PT3001-2) and Appendix D, Part B for instructions.

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Tet-On? 3G Inducible Expression Systems User Manual B. Protocol Summary
The following are the steps required to create a doxycycline-responsive cell line capable of inducible expression of your gene of interest (GOI) (see Figure 2). 1. Clone your gene of interest into a pTRE3G Vector using In-Fusion HD (Section V). 2. Pilot test Tet-based induction of your construct (Section VI). 3. Create a Tet3G-expressing stable cell line (Section VII). 4. Create and screen for a double-stable clone capable of high induction of your GOI (Section VIII).

Figure 2. Establishing the Tet-On 3G System in target cells. Target cells are transfected with the pCMV-Tet3G (or pEF1α-Tet3G) plasmid and selected with G418 to generate a stable Tet-On 3G cell line constitutively expressing Tet-On 3G transactivator. This cell line serves as the host for a PTRE3G-based expression vector, which is transfected into the Tet-On 3G cell line along with a linear selection marker (Hygr or Purr). After a second round of drug selection, a double-stable cell line is established which expresses high levels of the GOI in response to doxycycline (Dox).

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Tet-On? 3G Inducible Expression Systems User Manual

V.

Cloning Your Gene of Interest into a pTRE3G Vector using In-Fusion HD
We recommend using In-Fusion HD for all cloning. Follow the protocol outlined in the In-Fusion HD user manual (Type PT5162-1 in the keyword field at www.clontech.com/manuals).

Figure 3. The In-Fusion HD Single-Tube Cloning Protocol.

Depending on which pTRE3G vector you are using, the recommended linearization sites and forward/reverse primer designs are as follows:
Response Plasmid pTRE3G pTRE3G-IRES (MCSI) pTRE3G-IRES (MCSII) pTRE3G-mCherry pTRE3G-ZsGreen1 pTRE3G-BI (MCS-2) pTRE3G-BI (MCS-1) pTRE3G-BI-mCherry pTRE3G-BI-ZsGreen1 Linearize w/ SalI & BamHI SalI & EagI MluI & BamHI MluI & BamHI MluI & BamHI MluI BamHI BamHI BamHI Forward Primer* ccctcgtaaagtcgac 111 222 333 444 555 666 777 888 ccctcgtaaagtcgac 111 222 333 444 555 666 777 888 gccggatatcacgcgt 111 222 333 444 555 666 777 888 gccggatatcacgcgt 111 222 333 444 555 666 777 888 gccggatatcacgcgt 111 222 333 444 555 666 777 888 cgggggtaccacgcgt 111 222 333 444 555 666 777 888 atctccgcggggatcc 111 222 333 444 555 666 777 888 atctccgcggggatcc 111 222 333 444 555 666 777 888 atctccgcggggatcc 111 222 333 444 555 666 777 888 Reverse Primer** cagttacattggatcc SSS NNN NNN NNN NNN NNN NNN NNN ggagaggggccggccg SSS NNN NNN NNN NNN NNN NNN NNN cagttacattggatcc SSS NNN NNN NNN NNN NNN NNN NNN cagttacattggatcc SSS NNN NNN NNN NNN NNN NNN NNN cagttacattggatcc SSS NNN NNN NNN NNN NNN NNN NNN tatgctgcagacgcgt SSS NNN NNN NNN NNN NNN NNN NNN gcggatcgatggatcc SSS NNN NNN NNN NNN NNN NNN NNN gcggatcgatggatcc SSS NNN NNN NNN NNN NNN NNN NNN gcggatcgatggatcc SSS NNN NNN NNN NNN NNN NNN NNN

*111 = Start codon of your gene; 222 = 2nd codon of your gene; etc. **SSS = reverse compliment of the stop codon of your gene; NNN = reverse compliment of the end of your gene. NOTES: ? The Cla I restriction site in the pTRE3G and pTRE3G-IRES vectors is blocked by an overlapping dam methylation site. Therefore, in order to digest these vectors with Cla I, you must first propagate them in a dam– bacterial strain such as Clontech’s (dam–/dcm–) Stellar Competent Cells (Cat. No. 636764)—see Section III.H. ? For optimal expression of the downstream gene, the gene placed upstream of the IRES should not exceed 2.5 kb.

VI.

Pilot Testing Tet-Based Induction of Your Construct
Prior to establishing the double-stable Tet-On 3G cell line for your GOI, your pTRE3G construct should be tested for functionality. Transiently cotransfect your pTRE3G-GOI vector together with pCMV-Tet3G (in a 1:4 ratio for best inducibility) into an easy-to-transfect cell line such as HeLa or HEK 293, or your target cell line, and test for GOI induction with Dox. You will need an appropriate gene-specific assay to test for induction, such as: ? Western blot ? ? ? Northern blot qRT-PCR Gene-specific functional assay

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Tet-On? 3G Inducible Expression Systems User Manual
Alternatively you can perform a single vector transfection of pTRE3G-GOI into a newly created Tet-On 3G cell line (Section VII).

A.

Materials Required
1. pTRE3G Vector containing your gene of interest (Section V) 2. pCMV-Tet3G (or pEF1α-Tet3G) (Section II.C) 3. Host cell line 4. Xfect transfection reagent (Section III.F) 5. Doxycycline (1 mg/ml) (Section III.E) 6. Mammalian cell culture supplies (Section III.D) 7. Tet Approved FBS (Section III.A)

B.

Protocol
1. Cotransfect both the regulator and response plasmids into your target cells (in a 6-well plate) using Xfect transfection reagent. Follow the Xfect Protocol (Type PT5003-2 in the keyword field at www.clontech.com/manuals). ? ? Use 1 ?g of pCMV-Tet3G and 4 ?g of pTRE3G-GOI for each well (GOI = gene of interest). We recommend performing the test in duplicate with negative controls: 3 wells containing 100–1,000 ng/ml of Dox, and 3 wells without Dox.

Wells 1 & 2: 1 ?g pCMV-Tet3G and 4 ?g pTRE3G-GOI (no Dox) Wells 3 & 4: 1 ?g pCMV-Tet3G and 4 ?g pTRE3G-GOI (100–1,000 ng/ml Dox) Well 5: 1 ?g pCMV-Tet3G and 4 ?g pTRE3G empty (no Dox) Well 6: 1 ?g pCMV-Tet3G and 4 ?g pTRE3G empty (100–1,000 ng/ml Dox)

Figure 4. Transfection of the regulator and response plasmids into target cells in a 6-well plate.

2. After 24 hr, harvest the cell pellets from each well and compare induced expression levels to uninduced expression levels using a method appropriate for your GOI. NOTE: Because transiently transfected cells contain more copies of the TRE-containing plasmid than do stable cell lines, fold induction (ratio of maximal to basal GOI expression) levels are almost always lower in transient assays (e.g., by 10–100 fold) than in properly selected stable and doublestable clonal cell lines.

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Tet-On? 3G Inducible Expression Systems User Manual

VII.

Creating a Tet-On 3G-Expressing Stable Cell Line
NOTE: Skip Section VII if you have purchased a Tet-On 3G cell line. Instead, proceed to Section VIII. See the Tet Cell Lines Protocol-at-a-Glance (PT3001-2) and Appendix D, Part B for instructions on propagation and maintenance. The first step in establishing the Tet-On 3G System in your cells is creating a stable cell line that: (1) expresses the Tet-On 3G transactivator; (2) demonstrates high levels of induction from PTRE3G ; and (3) exhibits low basal expression from PTRE3G. This Tet-On 3G cell line will be frozen in aliquots and can be used to create individual inducible cell lines for all your genes of interest. Transfect using Xfect transfection reagent and select for colonies with G418 selection. In general, isolate enough colonies to be able to test at least 24 clones. Note that not all picked colonies will survive isolation and expansion. While it is possible to identify an optimal clone by screening fewer than 24 clones, our experience has shown that testing this many clones yields a high rate of success and will prevent significant delays. Your panel of 24 clones should then be screened by transient transfection with pTRE3G-Luc Control Vector to test for high induction and low basal expression using luciferase activity as a reporter. When you have identified a clone that demonstrates ideal induction characteristics, proceed to Section VIII to develop the double-stable Tet-On 3G inducible cell line. Be sure to freeze aliquots of your Tet-On 3G cell line(s) (Appendix D, Section A). NOTE: Working with mixed (polyclonal) populations of transfected cells, rather than selecting for single clones, can affect the consistency of induction due to the possible outgrowth of poorly inducing clones as the cells are passaged.

A.

Materials Required
1. pCMV-Tet3G (or pEF1α-Tet3G) (Section II.C) 2. pTRE3G-Luc Control Vector (Section II.B) 3. Host cell line 4. Xfect transfection reagent (Section III.F) 5. G418 (Section III.B) 6. Doxycycline (1 mg/ml) (Section III.E) 7. Mammalian cell culture supplies (Section III.D) 8. Tet Approved FBS (Section III.A)

B.

Protocol: Transfect and Select for 24 Independent Clones
1. Seed your target cells in a single well of a 6-well plate at a density sufficient to reach near confluence at 48 hr after transfection. Then transfect pCMV-Tet3G (or pEF1α-Tet3G) into your target cells using Xfect transfection reagent. 2. Follow the Xfect Protocol (PT5003-2 from www.clontech.com/manuals), except use 2 ?g of plasmid per well. NOTE: We use less DNA for stable transfections than required by the general Xfect protocol, to ensure that individual colonies are well-separated after G418 selection.

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Tet-On? 3G Inducible Expression Systems User Manual
3. After 48 hr, split the confluent well into 4 x 10 cm dishes (do not add G418 yet). 4. After an additional 48 hr, add G418 at the selection concentration that is optimal for your cell line. For most cell lines, this is usually 400–500 μg/ml (Section III.B). 5. Replace medium with fresh complete medium plus G418 every four days, or more often if necessary. 6. Cells that have not integrated the plasmid should begin to die after ~3–5 days. NOTE: Avoid passaging the cells a second time, since replating cells under selection may result in plates containing too many colonies for effective colony isolation (because individual colonies are not well-separated). 7. After ~2 weeks, G418-resistant colonies should begin to appear. 8. When the colonies are large enough to transfer, use cloning cylinders or disks to harvest (i.e., “pick”) large, healthy colonies, and transfer each into a separate well of a 24-well plate. Isolate as many clones as feasible, so that at least 24 clones are available for testing. Suspension cultures must be cloned using a limiting dilution technique (see Appendix C). 9. Culture the clones in a maintenance concentration of G418 (100–200 μg/ml). When confluent, split the cells from each well into three wells of a 6-well plate for testing and maintenance (Section VII.C). NOTE: You may wish to use TetR monoclonal antibody (Section III.I) to determine, via Western blot, which clones express the Tet-On 3G protein. However, Western analysis should not be used to substitute for a functional test for inducibility (Section VII.C), since the highest expressing Tet-On 3G clones often do not provide the highest fold inducibility.

C.

Protocol: Testing Your Tet-On 3G Clones for Induction
1. For each clone to be tested, seed 1/3 of the total amount of cells (Section VII.B, Step 9) into a single well of a 6-well plate. The cells in this “stock plate” may be propagated, depending upon the results of the screening assay. 2. Divide the remaining 2/3 of the cells between duplicate wells of a second 6-well plate. Allow the cells to adhere overnight, and transfect each well with 5 ?g of pTRE3G-Luc using Xfect transfection reagent. 3. After 4 hr, replace the culture medium with fresh medium and add Dox (100–1,000 ng/ml) to one of the duplicate wells, while leaving the second well Dox-free. 4. After 24 hr, assay for luciferase activity and calculate fold induction (e.g., +Dox RLU/–Dox RLU). 5. Select clones with the highest fold induction (ratio of maximal to basal gene expression) for propagation and further testing. NOTE: When testing clones via transient transfection, you can expect lower fold induction levels than in double-stable clones. This is because transiently transfected cells contain more copies of the TRE-containing plasmid than do stable cell lines. 6. Freeze stocks of each promising clone as soon as possible after expanding the culture (Appendix D).

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Tet-On? 3G Inducible Expression Systems User Manual

VIII. Creating & Screening for a Double-Stable Cell Line Capable of High Induction of your GOI
A. Materials Required
1. pTRE3G-GOI Vector (Section V) 2. Linear Hygromycin/Puromycin Marker (Section II.B) 3. Tet-On 3G Cell Line (Section VII) 4. Xfect transfection reagent (Section III.F) 5. G418 (Section III.B) 6. Doxycycline (1 mg/ml) (Section III.E) 7. Mammalian cell culture supplies (Section III.D) 8. Tet Approved FBS (Section III.A)

B.

Protocol: Creating a Double-Stable Tet-On 3G Inducible Cell Line
To generate a double-stable Tet-On 3G inducible cell line, cotransfect your customized pTRE-3G vector into your Tet-On 3G cell line along with a linear selection marker (Hygr or Purr). Select double-stable transfectants by screening for hygromycin or puromycin resistance, and inducibility. NOTE: Working with mixed (polyclonal) populations of transfected cells rather than selecting for single clones can affect the consistency of induction, due to the possible outgrowth of poorly inducing clones as the cells are passaged. Why use linear selection markers? See Appendix B. 1. Plate (seed) your Tet3G-expressing cell line in a single well of a 6-well plate at a density sufficient to reach near confluence at 48 hr after transfection. 2. Using Xfect transfection reagent (PT5003-2 from www.clontech.com/manuals), cotransfect the following: ? ? 2 ?g pTRE3G-GOI 100 ng Linear selection marker (puromycin or hygromycin)

NOTE: Always combine your customized pTRE3G vector and either the Linear Hygromycin Marker or the Linear Puromycin Marker at a ratio of 20:1 (i.e., use 20-fold less of the linear marker). 3. After 48 hr, split the confluent cells into 4 x 10 cm dishes (do not add the selective antibiotic yet). 4. After an additional 48 hr, add hygromycin or puromycin at the selection concentration that is optimal for your cell line (Section III.B). 5. Replace medium with fresh complete medium plus hygromycin (or puromycin) every four days, or more often if necessary.

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Tet-On? 3G Inducible Expression Systems User Manual
6. Cells that have not integrated the plasmid should begin to die after ~3–5 days. NOTE: Avoid passaging the cells a second time, since replating cells under selection may result in plates containing too many colonies for effective colony isolation (because individual colonies are not well-separated). 7. After ~2 weeks, drug-resistant colonies should begin to appear. 8. When the colonies are large enough to transfer, use cloning cylinders or disks to harvest (i.e. “pick”) large, healthy colonies, and transfer each into a separate well of a 24-well plate. Isolate as many clones as feasible, so that at least 24 clones are available for testing. Suspension cultures must be cloned using a limiting dilution technique (see Appendix C). 9. Culture the clones in maintenance concentrations of both G418 and hygromycin (or puromycin) (Section III.B). When confluent, split the cells from each well into three wells of a 6-well plate for testing and maintenance (Section VIII.C).

C.

Protocol: Screening Your Double-Stable Tet-On 3G Inducible Cell Lines
Test individual double-stable clones for expression of your GOI in the presence and absence of Dox (100–1,000 ng/ml). Choose clones that generate the highest maximal and lowest basal expression levels, i.e., the highest fold induction. 1. For each clone to be tested, seed 1/3 of the total amount of cells (see Section VIII.B, Step 9) into a single well of a 6-well plate. The cells in this “stock plate” may be propagated, depending upon the results of the inducibility assay. 2. Divide the remaining 2/3 of the cells between duplicate wells of a second 6-well plate. Add Dox (100–1,000 ng/ml) to one of the wells and incubate the cells for 48 hr. 3. Harvest the cells and use an assay specific for your GOI to compare induced to uninduced expression of your GOI. 4. Select clones with the highest fold induction for propagation and further testing. 5. Expand and freeze stocks of each promising clone as soon as possible (Appendix D). NOTE: Once you have chosen the best clone(s), you may choose to determine the minimal concentration of Dox that is required for high inducible expression and use that minimal concentration for all subsequent experiments. Remove the cells from one nearly confluent well (of a 6-well plate) and divide them among six wells of a 24-well plate. Titrate doxycycline concentrations across these 6 wells (e.g., 0, 1, 10, 50,100 & 1,000 ng/ml) and assay for induced expression after 24 hr).

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IX.

References

Clontech’s Tet Systems were developed in cooperation with Dr. Bujard and his colleagues at the Center for Molecular Biology in Heidelberg (ZMBH) and in Dr. Wolfgang Hillen’s laboratory at the University of Erlangen, Germany. Additional background information on Tet-regulated gene expression systems and an extensive bibliography are available at the website maintained by TET Systems: http://www.tetsystems.com (Please note that Clontech is not responsible for the information contained on this website.) Freshney, R.I. (2005). Culture of Animal Cells: A Manual of Basic Technique, 5th Edition (Wiley-Liss, Hoboken, NJ). Sambrook, J., Fritsch, E. F. & Maniatis, T., eds. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY). Gossen, M. & Bujard, H. (1992) Tight control of gene expression in mammalian cells by tetracycline responsive promoters. Proc. Natl. Acad. Sci. USA 89(12):5547–5551. Gossen, M., Freundlieb, S., Bender, G., Muller, G., Hillen, W. & Bujard, H. (1995) Transcriptional activation by tetracycline in mammalian cells. Science 268(5218):1766–1769. L?w, R., Heinz, N., Hampf, M., Bujard, H. & Gossen, M. (2010) Ameliorating the dynamic properties of the Tet system by altered minimal promoter design. Manuscript submitted for publication. Sambrook, J., Fritsch, E. F. & Maniatis, T., eds. (2001). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY). Urlinger, S., Baron, U., Thellmann, M., Hasan, M.T., Bujard, H. & Hillen, W. (2000) Exploring the sequence space for tetracycline-dependent transcriptional activators: Novel mutations yield expanded range and sensitivity. Proc. Natl. Acad. Sci. USA 97(14):7963–7968. Zhou, X., Vink, M., Klave, B., Berkhout, B. & Das, A. T. (2006) Optimization of the Tet-On system for regulated gene expression through viral evolution. Gene Ther. 13(19):1382–1390.

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Tet-On? 3G Inducible Expression Systems User Manual

X.

Troubleshooting
A. Low Fold Induction of Transient Expression
Solution We generally recommend a co-transient transfection A suboptimal ratio of cotransfected vector ratio of 1:4 for pCMV-Tet3G:pTRE3G-GOI vectors was used. (Section VI.B). Different vector ratios may result in different maximal/basal gene expression ratios. Cells were harvested and analyzed Harvest and analyze cells between 18–48 hr. too soon or too late. ? Optimize transfection protocol. Poor transfection efficiency ? Optimize density of cell plating; use at 60–90% confluency. ? Optimize passage number of target cells. Poor target cell viability ? Optimize culture conditions of target cells. ? Optimize tissue culture plasticware The FBS used in the cell culture medium contains tetracycline derivatives. Transiently transfected cells contain more copies of the TREcontaining plasmid than do stable cell lines. Use Clontech’s Tet System Approved FBS (Section III.A), which was functionally tested with Clontech’s double-stable CHO-AA8-Luc Tet-Off Control Cell Line. When testing clones via transient transfection, expect lower fold induction levels than in doublestable clones (sometimes only ~100-fold). Possible Explanation Description of Problem

Low fold induction (ratio of maximal to basal expression of the GOI)

B.

Low Fold Induction of Stable Expression
Possible Explanation Cellular sequences flanking the integrated TRE3G expression construct may affect GOI expression. Mixed cell population in the selected clone (see Section VIII.B Note). There is no direct correlation between the amount of expressed Tet-On 3G transactivator and induction efficiency. The appropriate antibiotics are missing from the cell culture medium. Mixed cell population in the selected clone (see Section VIII.B Note). Solution

Description of Problem Low fold induction of GOI expression in selected drugresistant double-stable cell clones. Low fold induction of GOI expression in selected drugresistant cell clones expressing Tet-On 3G transactivator, as detected by TetR Monoclonal Antibody Decrease in fold induction after several passages or Loss of inducibility after passaging of a (previously frozen) double-stable cell line.

Screen additional individual drug-resistant cell clones to ensure optimal fold induction.

Perform functional screening of selected drugresistant clones using pTRE3G-Luc (Section VII.C).

Maintain optimal antibiotic concentrations (Section III.B). Reselect the current cell line through single colony selection using selective concentrations of both antibiotics, and screen again with pTRE3G-Luc (Section VII.C).

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Tet-On? 3G Inducible Expression Systems User Manual C. Establishment of Stable Cell Lines
Possible Explanation The cells have not been recently passaged, so they remain wellattached to the plate surface even when they are dead. The antibiotic concentration which caused massive cell death when determining the appropriate dose via titration could be too high. Transfection was inefficient because cells used for transfection were of unsatisfactory quality, resulting in inefficient uptake of DNA during transfection. Inefficient transfection due to using the wrong ratio of Vector/Linear Selection Marker. Antibiotic was added too soon. Used wrong antibiotic concentration. ? Cells were not split and/or diluted correctly. ? Antibiotic was added too late. ? Transfected cells were passaged a second time after addition of antibiotic. Used wrong antibiotic concentration. Cells were not properly frozen. Cells were not properly thawed. Solution To determine the appropriate antibiotic concentration, use cells that have been split within the last 2–3 days.

Description of Problem Cells do not die at the high antibiotic concentration established via titration in Section III.B There are no surviving cells after transfection/cotransfection with a drug-resistant expression cassette at the antibiotic concentration determined to be optimal in Section III.B

Use a lower antibiotic concentration for selection of stably transfected cell clones.

Low number of drug resistant clones

Use cells for transfection at passages no higher than 15–17 since defrosting, and no older than 2–3 days since the last split. Passage cells 3–4 times after defrosting to allow a complete cell recovery prior to transfection experiments. Check the ratio of Vector/Linear Selection Marker. Retransfect Vector/Linear Selection Marker at a ratio of 20:1 (Section VIII.B). See protocols in Sections VII.B & VIII.B. See Section III.B

Too many colonies for effective colony isolation (individual colonies are not well-separated)

See protocols in Sections VII.B & VIII.B.

Poor cell viability

See Section III.B See Appendix D, Section A. See Appendix D, Section B.

D.

Detection and Inhibition of Expression
Possible Explanation Low sensitivity of detection method. Depending on the stability of the protein, it may persist in the cell in the absence of gene induction and de novo synthesis of GOI mRNA. Fluorescent proteins tend to have long half-lives. Solution Check sensitivity of primary and secondary antibodies. Analyze GOI expression by qRTPCR, using different sets of primers to ensure optimal detection of GOI expression. Upon degradation, GOI/Fluorescent Protein expression will not be detectable in cells in the absence of induction. For faster degradation of an inducible GOI, use pTRE-Cycle Vectors (see www.clontech.com). Wash cells three times with PBS, followed by trypsinization and replating in fresh medium supplemented with Clontech’s Tet System Approved FBS. If trypsinization is undesirable, wash cells three times with medium and three times with PBS, then replace with fresh medium supplemented with Tet System Approved FBS.

Description of Problem No detectable GOI expression by Western Blot.

Continuous GOI/Fluorescent Protein expression after the removal of doxycycline

Doxycyline was not completely removed from the cell culture medium.

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Appendix A: Tet-On 3G Systems Vector Information
The Tet-On 3G Inducible Expression Systems (Section II) each contain one of two possible regulator plasmids (Figure 5) and one of four possible response plasmids (Figures 6 & 7), as well as a pTRE3G-Luc control response plasmid (Figure 8). For complete descriptions of the vectors provided with each system, refer to the enclosed Certificate of Analysis, which is also available at www.clontech.com

Figure 5. pCMV-Tet3G Vector and pEF1α-Tet3G Vector Maps.

Figure 6. pTRE3G Vector and pTRE3G-IRES Vector Maps.

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Figure 7. pTRE3G-mCherry Vector and pTRE3G-ZsGreen1 Vector Maps.

Figure 8. pTRE3G-Luc Control Vector Map.

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Figure 9. pTRE3G-BI-Luc Control Vector and pTRE3G-BI Vector Maps.

Figure 10. pTRE3G-BI-mCherry Vector and pTRE3G-BI-ZsGreen1 Vector Maps.

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Tet-On? 3G Inducible Expression Systems User Manual

Appendix B: Why Use a Linear Selection Marker?
Linear selection markers are short, purified linear DNA fragments that consist of the marker gene (Hygr or Purr), an SV40 promoter, and the SV40 polyadenylation signal. Use of a linear selection marker allows you to screen fewer clones to obtain your desired clone; plus, you'll observe a higher fold induction in the clones that you select. Why is this? because there is less interference with basal expression of the gene of interest from the promoter of a cotransfected linear selection marker than would result from the promoter of a selection marker present on the pTRE3GGOI plasmid itself. This is due to the fact that stable integration of plasmids usually results in co-integration of multiple copies of that plasmid at a single locus. If pTRE3G were supplied with a constitutive selectable marker included on the plasmid backbone (i.e., a constitutive promoter at an automatic 1:1 ratio to the TRE promoter), the constitutive promoter used for the selection marker could affect basal expression in many of the clones by a combination of: ? ? its juxtaposition with the TRE in one or more of the tandem integrations or the recruitment of a high concentration of endogenous transcription factors to the region

However, since the linear selection markers are cotransfected at a decreased ratio of 1:20 relative to the pTREG-GOI plasmid (i.e., 20-fold less of the linear marker), these types of interference are less likely to occur.

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Appendix C: Selecting Stable Clones via Limited Dilution of Suspension Cells
To avoid creating a cell line containing a mixture of clones, suspension cells must be selected using a limited dilution technique. The following protocol allows you to dilute stably transfected cells in a manner ensuring that only one stable cell clone is seeded per well in a 96-well plate—and then use that clone to test for inducible expression.

A.

Protocol
1. Seed one well of a 6-well plate with 1–1.5 x 106 cells in 3 ml of complete growth medium. 2. Using Xfect transfection reagent, transfect these cells with 5 ?g of your plasmid according to the Xfect protocol (type PT5003-2 in the keyword field at www.clontech.com/manuals). 3. 48 hr after transfection, centrifuge at 1,100 rpm to harvest the cells, and resuspend them in 6 ml of medium in a T25 flask containing the appropriate antibiotic to select for stable integrants (e.g., use G418 to select for pCMV-Tet3G or pEF1?-Tet3G). 4. Allow the cells to grow for 1 week. 5. Dilute the cells from Step 4 to 1 cell per well in a 96-well plate as follows: a. Dilute a 100 ?l aliquot of the cells in 2 ml of complete medium (1/20 stock dilution). b. Set up four vials containing 5 ml of complete growth medium. From the 1/20 stock dilution created in Step 5.a, add: i. 10 ?l to Vial 1 ii. 20 ?l to Vial 2 iii. 30 ?l to Vial 3 iv. 40 ?l to Vial 4 c. Mix well. d. From Vial 1, add one 50 ?l aliquot per well to each well of a 96-well plate. Repeat this process for Vials 2–4 on separate 96-well plates (four plates total—one for each vial). 6. Allow the cells on each of the four 96-well plates to grow until growth is visible in half of the wells on one of the plates. 7. Choose 24 clones only from the plate that shows growth in approximately half of its wells. Expand each of these clones to fill one well of a 24-well plate and then one well of a 6-well plate. NOTE: If one of the 96-well plates shows growth in only half of its wells, this means that on average there was less than one cell per well on that plate when they were seeded (Step 5.d), so the cells in the wells that show growth are likely to have been derived from a single cell clone. 8. When each of the 24 clones in Step 7 has grown sufficiently to fill 3 wells of a 6-well plate, maintain the cells from one well as the reference stock, and test the cells in the other two wells for inducible expression with and without Dox (see Sections VII.C and VIII.C).

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Appendix D: Preparing and Handling Tet-On 3G Cell Line Stocks
A. Protocol: Freezing Tet-On 3G Cell Line Stocks
Once you have created and tested your Tet-On 3G cell line, you must prepare multiple frozen aliquots to ensure a renewable source of cells, according to the following protocol: 9. Expand your cells to multiple 10 cm dishes or T75 flasks. 10. Trypsinize and pool all of the cells, then count the cells using a hemocytometer. 11. Centrifuge the cells at 100 x g for 5 min. Aspirate the supernatant. 12. Resuspend the pellet at a density of at least 1–2 x 106 cells/ml in freezing medium. Freezing medium can be purchased from Sigma (Cat. Nos. C6164 & C6039), or use 70–90% FBS, 0–20% medium (without selective antibiotics), and 10% DMSO. 13. Dispense 1 ml aliquots into sterile cryovials and freeze slowly (1°C per min). For this purpose, you can place the vials in Nalgene cryo-containers (Nalgene 6. Cat. No. 5100) and freeze at –80°C overnight. Alternatively, place vials in a thick-walled styrofoam container at –20°C for 1–2 hr. Transfer to –80°C and freeze overnight. 14. The next day, remove the vials from the cryo-containers or styrofoam containers, and place in liquid nitrogen storage or an ultra-low temperature freezer (–150°C) for storage. 15. Two or more weeks later, plate a vial of frozen cells to confirm viability.

B.

Protocol: Thawing Tet-On 3G Cell Line Frozen Stocks
To prevent osmotic shock and maximize cell survival, use the following procedure to start a new culture from frozen cells: 1. Thaw the vial of cells rapidly in a 37°C water bath with gentle agitation. Immediately upon thawing, wipe the outside of the vial with 70% ethanol. All of the operations from this point on should be carried out in a laminar flow tissue culture hood under strict aseptic conditions. 2. Unscrew the top of the vial slowly and, using a pipet, transfer the contents of the vial to a 15 ml conical centrifuge tube containing 1 ml of prewarmed medium (without selective antibiotics such as G418). Mix gently. 3. Slowly add an additional 4 ml of fresh, prewarmed medium to the tube and mix gently. 4. Add an additional 5 ml of prewarmed medium to the tube and mix gently. 5. Centrifuge at 100 x g for 5 min, carefully aspirate the supernatant, and GENTLY resuspend the cells in complete medium without selective antibiotics. (This method removes the cryopreservative and can be beneficial when resuspending in small volumes. However, be sure to treat the cells gently to prevent damaging fragile cell membranes.)

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6. Mix the cell suspension thoroughly and add to a suitable culture vessel. Gently rock or swirl the dish/flask to distribute the cells evenly over the growth surface and place in a 37°C humidified incubator (5–10% CO2 as appropriate) for 24 hr. NOTE: For some loosely adherent cells (e.g. HEK 293-based cell lines), we recommend using collagen-coated plates to aid attachment after thawing. For suspension cultures, suspend cells at a density of no less than 2 x 105 cells/ml. 7. The next day, examine the cells under a microscope. If the cells are well-attached and confluent, they can be passaged for use. If the majority of cells are not well-attached, continue culturing for another 24 hr. NOTE: Note: For some loosely adherent cell lines (e.g., HEK 293-based cell lines), complete attachment of newly thawed cultures may require up to 48 hr. 8. Expand the culture as needed. The appropriate selective antibiotic(s) should be added to the medium after 48–72 hr in culture. Maintain stable and double-stable Tet Cell Lines in complete culture medium containing a maintenance concentration G418 and/or hygromycin (or puromycin), as appropriate (Section III.B).

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