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Clontech Laboratories, Inc. Retro-X Tet-One Inducible Expression System User Manual Cat. Nos , (010814) Clontech Laboratories, Inc. A Takara Bio Company 1290 Terra Bella Avenue, Mountain
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Clontech Laboratories, Inc. Retro-X Tet-One Inducible Expression System User Manual Cat. Nos , (010814) Clontech Laboratories, Inc. A Takara Bio Company 1290 Terra Bella Avenue, Mountain View, CA 94043, USA U.S. Technical Support: United States/Canada Asia Pacific Europe +33.(0) Japan +81.(0) Page 1 of 28 Table of Contents I. Introduction... 3 II. List of Components... 5 III. Additional Materials Required... 6 IV. Protocol Overview... 9 V. Cloning Your Gene of Interest into a pretrox-tetone Vector using In-Fusion HD VI. Pilot Testing Tet-Based Induction of Your Construct A. Materials Required B. Protocol VII. Producing Retrovirus from the Retro-X Vectors A. General Considerations B. Protocol: Packaging Retroviral Vectors Using Xfect Transfection Reagent VIII. Retrovirus Titration A. Titrating Your Retroviral Supernatants by qrt-pcr B. Protocol: Determining Viral Titer Using Antibiotic Selection IX. Transducing Target Cells with the Tet-One Retroviruses A. Summary B. Protocol: Transducing a Mixed Population without Clonal Selection C. Protocol: Screening Single Clones Using Puromycin Selection D. Protocol: Screening Single Clones Using Limiting Dilution E. Protocol: Testing Your Tet-One Clones for Induction X. References Appendix A. Troubleshooting Guide Appendix B: Retro-X Tet-One System Vector Information Appendix C: Preparing and Handling Cell Line Stocks Page 2 of 28 Table of Figures Figure 1. The Tet-On 3G and Tet-One Systems allow inducible gene expression in the presence of Dox Figure 2. Establishing an inducible expression system in target cells with Retro-X Tet-One Figure 3. The In-Fusion HD Single-Tube Cloning Protocol Figure 4. Transfection of the pretrox-tetone vectors into target cells in a 6-well plate Figure 5. Flowchart of procedures for titering retrovirus supernatants with the Retro-X qrt-pcr Titration Kit Figure 6. pretrox-tetone Vector Map Figure 7. pretrox-tetone-luc Control Vector Map Figure 8. pretrox-tetone-puro Vector Map Figure 9. pretrox-tetone-puro-luc Control Vector Map Table of Tables Table 1. Recommended Antibiotic Concentrations for Selecting & Maintaining Stable Cell Lines... 6 Table 2. Tropisms associated with Commonly Used Retroviral Envelopes Table 3. Troubleshooting Guide for the Retro-X Tet-One Inducible Expression System I. Introduction A. Summary The Tet-One Systems are inducible gene expression systems for mammalian cells that contain all the necessary components in a single plasmid, lentiviral, or retroviral vector. After transfecting target cells with plasmid (Tet-One Systems), or transducing them with retrovirus (Retro-X Tet-One systems) or lentivirus (Lenti-X Tet-One Systems), the cells will express the Tet-On 3G transactivator protein and contain a gene of interest (GOI) under the tight control of a TRE3G promoter (P TRE3GS ). This manual describes the retrovirus-based Retro-X Tet-One Inducible Expression System (Cat. No ) and Retro-X Tet-One Inducible Expression System (Puro) (Cat. No ). Using these systems, your target cells 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 and Tet-One Systems allow inducible gene expression in the presence of Dox. Page 3 of 28 B. Elements of Retro-X Tet-One Systems 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). P TRE3GS Inducible Promoter The inducible promoter P TRE3G provides for very low basal expression and high maximal expression after induction (Loew et. al., 2010). It consists of 7 repeats of a 19 bp tet operator sequence located upstream of a minimal CMV promoter. P TRE3GS is a version of P TRE3G that was modified for higher performance in a single vector context. In the presence of Dox, Tet-On 3G binds specifically to P TRE3GS and activates transcription of the downstream GOI. P TRE3GS lacks binding sites for endogenous mammalian transcription factors, so it is virtually silent in the absence of induction. Tet-One Systems All-in-One Design Before the Tet-One Systems were developed, Clontech s Tet-On and Tet-Off products all required two separate vectors to introduce the transactivator protein and the inducible promoter controlling your gene of interest, respectively, into your target cells. The Tet-One Systems provide both of these components on a single vector. The Tet-On 3G transactivator is expressed in the forward direction from the human phosphoglycerate kinase 1 promoter, and the cloned gene of interest is expressed from the P TRE3GS promoter in the reverse orientation. Compared to the two-vector Tet-On 3G Systems, all previously published all-in-one vectors have shown a low signal-to-noise ratio, typically providing only fold induced expression, even in selected clones. Clontech s Tet-One Systems are based on an all-in-one design that has shown up to 25,000-fold induction (Heinz et. al,. 2011). Retro-X Universal Packaging System The Retro-X Universal Packaging System, provided with the Retro-X Tet-One Inducible Expression System and the Retro-X Tet-One Inducible Expression System (Puro), is our premium retroviral packaging system, featuring the high titer GP2-293 packaging cell line. All four commonly used envelopes are supplied on separate vectors, including VSV-G, eco, ampho and 10A1, to allow you to choose the tropism that is most appropriate for your target cells. The envelope vector is cotransfected with your retroviral expression vector, and high titers of pantropic, ecotropic, amphotropic, or dualtropic virions can be obtained in less than 48 hr (see Table 2). C. Doxycycline Doxycycline is a synthetic tetracycline derivative that is the effector molecule for all 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 P TRE3GS promoter (Figure 1). The Dox concentrations required for induction 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 and Tet-One Systems respond well only to doxycycline, and not to tetracycline (Gossen & Bujard, 1995). The half-life of Dox in cell culture medium is 24 hr. To maintain continuous inducible GOI expression in cell culture, the medium should be replenished with Dox every 48 hr. Page 4 of 28 II. List of Components Store the GP2-293 Packaging Cell Line at 196 C and all other components at 20 C. Retro-X Tet-One Inducible Expression System (Cat. No ) 1 each pretrox-tetone Vector Set (Cat. No ; not sold separately) 20 μl 20 μl pretrox-tetone Vector (500 ng/µl) pretrox-tetone-luc Control Vector (500 ng /µl) 1 each Retro-X Universal Packaging Vector Set (Cat. No ; not sold separately) 20 μl 20 μl 20 μl 20 μl 20 μl p10a1 Vector (500 ng/µl) pampho Vector (500 ng /µl) peco Vector (500 ng /µl) pvsv-g Vector (500 ng /µl) pqclin Retroviral Vector (500 ng /µl)) 1 ml GP2-293 Packaging Cell Line (2 x 10 6 cells/ml) (Cat. No ; not sold separately) 100 rxns Xfect Transfection Reagent (Cat. No ) 2 tubes 2 tubes Xfect Polymer (75 μl each) Xfect Reaction Buffer (12 ml each) 50 ml Tet System Approved FBS, US-Sourced (Cat. No ) Retro-X Tet-One Inducible Expression System (Puro) (Cat. No ) 1 each pretrox-tetone-puro Vector Set (Cat. No ; not sold separately) 20 μl 20 μl pretrox-tetone-puro Vector (500 ng/µl) pretrox-tetone-puro-luc Control Vector (500 ng /µl) 1 each Retro-X Universal Packaging Vector Set (Cat. No ; not sold separately) 20 μl 20 μl 20 μl 20 μl 20 μl p10a1 Vector (500 ng/µl) pampho Vector (500 ng /µl) peco Vector (500 ng /µl) pvsv-g Vector (500 ng /µl) pqclin Retroviral Vector (500 ng /µl) 1 ml GP2-293 Packaging Cell Line (2 x 10 6 cells/ml) (Cat. No ; not sold separately) 100 rxns Xfect Transfection Reagent (Cat. No ) 2 tubes 2 tubes Xfect Polymer (75 μl each) Xfect Reaction Buffer (12 ml each) 50 ml Tet System Approved FBS, US-Sourced (Cat. No ) NOTE: The only difference between the two systems is the presence of a puromycin resistance cassette in the pretrox-tetone-puro Vector to allow for selection of stable clones using antibiotic selection (Section IX.C). pretrox-tetone does not contain a selection marker gene and as a result allows for a larger transgene to be cloned (up to 3.1 kb, compared to ~2.1 kb for pretrox -TetOne-Puro). Transduced clones created using pretrox -TetOne can instead be isolated by limiting dilution (Section IX.D). Page 5 of 28 III. Additional Materials Required The following reagents are required but not supplied. 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. 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) B. Antibiotic for Selecting Stable Cell Lines The RetroX-TetOne-Puro Vector contains a puromycin resistance marker for selection of stable clones or populations (Section IX.C). Use the following recommended puromycin concentrations: Table 1. Recommended Antibiotic Concentrations for Selecting & Maintaining Stable Cell Lines Recommended Concentration (µg/ml) Cat. No. Antibiotic Selecting Colonies 1 Maintenance Puromycin (100 mg) Puromycin (25 mg) 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. pretrox-tetone does not contain a selection marker. However, clones can instead be isolated using limiting dilution (Section IX.D). C. Mammalian Cell Culture Supplies Medium for GP2-293 Cells: 90% Dulbecco's Modified Eagle's Medium (DMEM) with high glucose (4.5 g/l), 4 mm L-glutamine, and sodium bicarbonate (Sigma-Aldrich, D5796); 10% Fetal Bovine Serum (FBS); 100 units/ml penicillin G sodium & 100 μg/ml streptomycin sulfate. 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 stable Tet-One and GP2-293 cell lines. 6-well, 12-well, 24-well, and 96-well cell culture plates; 10 cm cell culture dishes Page 6 of 28 D. Retroviral Titer Determination For accurate and consistent transductions, we highly recommend titrating your retroviral stocks. Visit for details. Cat. No. Retroviral Titration Technology Retro-X qrt-pcr Titration Kit (200 rxns) E. Retrovirus Concentration Cat. No. Concentrator Retro-X Concentrator (100 ml) Retro-X Concentrator (500 ml) Use Retro-X Concentrator to simply increase your available titer up to 100-fold or reduce sample volume, without ultracentrifugation visit for details. F. Transduction Enhancers Use Polybrene (hexadimethrine bromide; Sigma-Aldrich, No. H9268), Lenti-X Accelerator (see below), or RetroNectin (see below). Lenti-X Accelerator is a magnetic bead-based technology designed to accelerate lentiviral and retroviral transduction experiments; visit for details. RetroNectin is a multivalent molecule that simultaneously binds virus particles and cell surface proteins, maximizing cell-virus contact. RetroNectin, in particular, is recommended for increasing the transduction efficiency of suspension cells and stem cells; visit for details. Cat. No. Transduction Enhancer Size Lenti-X Accelerator 400 µl Lenti-X Accelerator 1,000 µl Lenti-X Accelerator Starter Kit each T110A RetroNectin Precoated Dish 10 dishes T100B RetroNectin Recombinant Human Fibronectin Fragment 2.5 mg T100A RetroNectin Recombinant Human Fibronectin Fragment 0.5 mg G. Doxycycline 5 g Doxycycline (Cat. No ) Dilute to 1 mg/ml in double distilled H 2 O. Filter sterilize, aliquot, and store at 20 C in the dark. Use within one year. H. Xfect Transfection Reagent Xfect provides high transfection efficiency for most commonly used cell types, including GP2-293 cells. Cat. No. Transfection Reagent Xfect Transfection Reagent (100 rxns) Xfect Transfection Reagent (300 rxns) Page 7 of 28 I. In-Fusion HD Cloning System In-Fusion is a revolutionary technology that greatly simplifies cloning. For more information, visit Cat. No. In-Fusion Cloning Kit In-Fusion HD Cloning Plus (10 rxns) In-Fusion HD Cloning Plus (50 rxns) In-Fusion HD Cloning Plus (100 rxns) J. Stellar Competent Cells Stellar Competent Cells are recommended by Clontech for cloning of lentiviral and retroviral vectors. Propagation of vectors containing repeat sequences such as viral LTRs using other strains of E. coli may result in plasmid rearrangements. Stellar Competent Cells are sold separately and provided with all In- Fusion HD Cloning Systems. Cat. No. Competent Cells Stellar Competent Cells (10 x 100 µl) Stellar Competent Cells (50 x 100 µl) K. 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. We do not recommend using the TetR Monoclonal Antibody to screen clones. Cat. No. Antibody TetR Monoclonal Antibody (Clone 9G9) (40 µg) TetR Monoclonal Antibody (Clone 9G9) (200 µg) L. Plasmid Purification (Transfection-Grade) Cat. No. Product Size NucleoBond Xtra Midi Plus 10 preps NucleoBond Xtra Maxi Plus 10 preps NucleoBond Xtra Midi EF Plus 10 preps NucleoBond Xtra Maxi EF Plus 10 preps M. Luciferase Assay and Luminometer These items are required when using the pretrox-tre3g-luc Vector or the pretrox-tre3g-luc-puro Vector as a control to test for induction (Section VI.B). Use any standard firefly luciferase assay system and luminometer. Page 8 of 28 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 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 (Wiley-Liss, Hoboken, NJ). B. Safety Guidelines for Working with Retroviruses The protocols in this User Manual require the production, handling, and storage of infectious retrovirus. It is imperative to fully understand the potential hazards of, and necessary precautions for, the laboratory use of retroviruses. The National Institute of Health and Center for Disease Control have designated retroviruses such as Moloney murine leukemia virus (MMLV) as Level 2 organisms. This requires the maintenance of a Biosafety Level 2 facility for work involving this virus and others like it. MMLV does not naturally infect human cells; however, virus packaged from the MMLV-based vectors described here is capable of infecting human cells. The viral supernatants produced by these retroviral systems could, depending on your retroviral insert, contain potentially hazardous recombinant virus. Similar vectors have been approved for human gene therapy trials, attesting to their potential ability to express genes in vivo. IMPORTANT: For these reasons, due caution must be exercised in the production and handling of any recombinant retrovirus. The user is strongly advised not to create retroviruses capable of expressing known oncogenes in amphotropic or polytropic host range viruses. For more information on Biosafety Level 2 agents and practices, download the following reference: Biosafety in Microbiological and Biomedical Laboratories (BMBL), Fifth Edition (Revised December 2009) HHS Pub. No. (CDC) U.S. Department of Health and Human Services Public Health Service, Centers for Disease Control and Prevention, and NIH. Available on the web at Biosafety Level 2: The following information is a brief description of Biosafety Level 2. It is neither detailed nor complete. Details of the practices, safety equipment, and facilities that combine to produce a Biosafety Level 2 are available in the above publication. If possible, observe and learn the practices described below from someone who has experience working with retroviruses. Page 9 of 28 Summary of Biosafety Level 2: Practices: Standard microbiological practices Limited access to work area Biohazard warning signs posted Minimize production of aerosols Decontaminate potentially infectious wastes before disposal Use precautions with sharps (e.g., syringes, blades) Biosafety manual defining any needed waste decontamination or medical surveillance policies Safety equipment: Biological Safety Cabinet, preferably a Class II BSC/laminar flow hood (with a HEPA microfilter) used for all manipulations of agents that cause splashes or aerosols of infectious materials; exhaust air is unrecirculated PPE: protective laboratory coats, gloves, face protection as needed Facilities: Autoclave available for waste decontamination Chemical disinfectants available for spills Page 10 of 28 C. Protocol Summary The following are the steps required to create a doxycycline-inducible expression system using retrovirus (see Figure 2). 1. Clone your gene of interest into the pretrox-tetone Vector using In-Fusion HD (Section V). 2. Pilot test Tet-based induction of your construct using transient transfection (Section VI). 3. Produce retroviral supernatants using the Retro-X Universal Packaging System (Section VII). 4. Transduce your target cells with TetOne virus (Section IX). Figure 2. Establishing an inducible expression system in target cells with Retro-X Tet-One. The pretrox-tetone plasmid containing your gene of interest and an envelope vector are cotransfected into GP2-293 target cell lines, and used to generate a high-titer retroviral supernatant (Section VII). The retroviral supernatant is used to transduce your target cells (Section IX). Clones are then selected, expanded, and screened for doxycycline-inducible expression of your gene of interest. Page 11 of 28 V. Cloning Your Gene of Interest into a pretrox-tetone 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 at NOTE: Stellar Competent Cells (Section III.J) are recommended by Clontech for cloning of lentiviral and retroviral vectors. Propagation of vectors containing repeat sequences such as viral LTRs using other strains of E. coli may result in plasmid rearrangements. Stellar Competent Cells are provided with all In-Fusion HD Cloning Systems. Figure 3. The In-Fusion HD Single-Tube Cloning Protocol. The recommended linearization sites and forward/reverse primer designs are as follows: Vector Linearize with Forward Primer* Reverse Primer** pretrox-tetone EcoRI & BamHI CCCTCGTAAAGAATTC GAGGTGGTCTGGATCCSSS NNN NNN NNN NNN NNN NNN NNN pretrox -TetOne-Puro EcoRI & BamHI CCCTCGTAAAGAATTC GAGGTGGTCTGGATCC SSS NNN NNN NNN NNN NNN NNN NNN *111 = Start codon of your gene; 222 = 2nd codon of your gene; etc. **SSS = reverse complement of the stop codon of your gene; NNN = reverse complement of the end of your gene. Page 12 of 28 VI. Pilot Testing Tet-Based Induction of Your Construct Prior to retrovirus production, your pretrox-tetone or pretrox-tetone-puro construct should be tested for functionality by plasmid transfection. Transiently
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