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BD Tet-Off and Tet-On Gene Expression System

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1. Tet Off Cell Lines Protocol 4 PT3001 1 Version PR33678 Doxycycline a derivative of Tcthat is the preferred effector substance for Tet experiments A Tet Off or Tet On cell line that has been stably transfected with pTRE2 Gene X construct Gene X is induced by the removal for Tet Off or addition for Tet On of Dox from the media The gene of interest cloned into the Response Plasmid The complete immediate early promoter of cytomegalovirus This is a proven strong promoter in many mammalian cell types Theminimalimmediate early CMV promoter This promoter lacksthe strong CMV enhancer andistherefore silentin the absence of binding of tTA or rtTA to the TRE Analtered minimal immediate early CMV promoter This promoter is used in the pTRE Tight vector series The compound promoter in pTRE and related vectors that consists of the TRE element located just upstream of The compound promoter in the pTRE Tight vectors that consists of the TRE element located just upstream of Princuva The plasmid that encodes the hybrid regulatory protein or rtTA in a Tet Off or Tet On System i e pTet Off or p Tet A pTRE derived plasmid that expresses a gene of interest from the Promy 1 promoter A pTRE derived plasmid can be used in both Tet Off and Tet On systems The reverse Tet repressor In E coli rTetR binds specifically to tetO and blocks transcription of the fet operon in the presenc
2. Hweg L 0 9 ON euou 6 3 1d Hweg 92 PUIH ee euou d4do3 446 3 19 LO SO i uo oes 8862 euou ddo32p ddo32p au1d 8661 1219 X N ddo32p au1d 9 9 Hweg NHX9 3H1d papiaoid 871 ee S S euou eseJejlon NHX9 onT NHX9 3u 1d Zs A H093 91992 HI eS X uiejoJd NHX9 19 A H093 9199 uloAwoiBAy 9 Ld 8 0 0 E 4099 ge Hweg 9 euou X ut9104d NHX9 NHX9 3u 1d 918 819 9 9 Hweg 68vG euou onT vH 3u 1d cS 8023 9199 zs uloAwound X ulajoid WH 1 8023 9199 yg uloAwoiBAy X ulejoJd vyH VH cD uzau 1d 100 LW 1009A ge Hweg 8 t euou XxulejoJd vH VH 3u1d s awAzua 92u9J9Jo1 59215 0011211891 215 o qeioojos jueuibej4 onsouDeiq uelewweyy NOILVINYOANI HOLO3A SINALSAS 131 Ill 318V L Protocol 4 PT3001 1 Version PR33678 www bdbiosciences com BD Biosciences Clontech 40 Tet Systems User Manual inued ion cont Vector Informati Appendix A 18023 40S euou 6AH x1d d
3. Cycljip LacZ GAPDH We e Figure 1 Inducible on off control of gene expression in the Tet Systems Panel Double stable lines were developed by stably transfecting HeLa Tet Off or HeLa Tet On cells with a plasmid containing coli lacZ under control of the Tet response element TRE Cells were cultured 1 ug ml Dox For Northern analysis 10 ug of total RNA was loaded per lane and the blot was hybridized simultaneously with probes to 2 and the GAPDH housekeeping gene Gossen et al 1995 reprinted with permission of the author Panel B HeLa S3 Tet Off cells were stably transfected with a plasmid expressing Bcl 2 under control of the TRE and grown in the presence of the indicated amounts of Tc A Western blot containing 100 ug of total protein from each condition was probed with human Bcl 2 specific and human cyclin B1 specific mouse monoclonal antibodies Based on scanning densitometry removal of Tc gave 100 fold induction of Bcl 2 For details see Yin amp Schimke 1995 BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 4 Version PR33678 Tet Systems User Manual Introduction continued See Appendix A or the Vector Information Packets provided for maps and detailed information on the Tet System Vectors For a complete list of Tet Systems references visitour website at www bdbiosciences com clontech B The BD Tet Off and Tet O
4. 20 C in the dark Use within two months For luciferase assays Use any standard luciferase assay system We recommend our Luciferase Reporter Assay Kit 631714 For PCR confirmation of integrated plasmids optional If you wish to confirm the presence of integrated plasmids in clonal hygromycin puromycin or neomycin resistant cell lines you will need to design PCR primers that amplify a portion of the appropriate regulator or response plasmid Protocol 4 PT3001 1 www bdbiosciences com BD Biosciences Clontech Version PR33678 17 Tet Systems User Manual V Plasmid Manipulations A Propagation of Vector Plasmids 1 Transform each of the plasmids provided in this kit into a suitable E coli host strain e g DH5a to ensure that you have a renewable source of DNA Tet vectors are low copy number so use chloramphenicol amplification to increase plasmid yields 2 You will need to perform large scale plasmid preparations of any plasmid that will be introduced into mammalian cells To ensure the purity of the DNA prepare transfection grade plasmid by purification NucleoBondG column Visit www bdbiosciences com clontech for complete product information B Generating Your Gene Specific Expression Vector Generate your pTRE Gene X construct using standard molecular biology techniques as described below For more detailed information see Sambrook et al 1989 1 Purify the Gene X fragment by any standar
5. activation domain ptTA2 3 4 7 1 kb Mig y 1987 Tolerated Relative Stable level of transient regulation VP16 activation domain activator activation 96 factor pTet Off 1X 100 22x10 ptTA2 3X 98 nd 5 Scal Bsal ptTA3 5 39 15x10 3686 3267 ptTA4 G F Y 9X 14 44 x 104 Figure 17 VP16 Minimal Domain Vectors The three vectors differ in the sequence of their VP16 activation domains The letters in the first column of Panel B indicate the amino acid at the key functional position of a 13 amino acid repeat that composes the minimal domains The rest of the vector is identical to pTet Off The activation domain from each vector is tolerated at different levels and causes activation at different levels relative to pTet Off Panel B not determined Protocol 4 PT3001 1 www bdbiosciences com BD Biosciences Clontech Version PR33678 49 Tet Systems User Manual Appendix A Vector Information continued The pTet tTS Vector The pTet tTS Vector 631011 is designed for use with the Tet On System It is not suitable foruse with the Tet Off System pTet tTS prevents unregulated gene expression in the absence of Dox Clontechniques April 1999 It expresses the tetracycline controlled transcriptional silencer tTS which is a fusion of TetR and the KRAB AB domain of the Kid 1 protein Freundlieb et a 1999 Witzgall et al 1994 In the absence of Dox tTS binds the tetO sequence in the
6. which license specifically excludes the right to sell or otherwise transfer the Tet Technology or its component parts to third parties In accepting this license all users acknowledge that the Tet Technology is experimen tal in nature Abbott makes no warranties express or implied or of any kind and hereby disclaims any warranties representations or guarantees of any kinds as to the Tet Technology patents or products All others are invited to request a license from Abbott prior to purchasing these reagents or using them for any purpose BD Biosciences Clontech is required by its licensing agreement to submit a report of all purchasers of the Tet controllable expression systems to Abbott For license information please contact Abbott Bioresearch Center 100 Research Drive Worcester MA 01605 4314 U S A Fax 508 755 8506 Use of BD Biosciences Clontech s Living Colors products containing DNA sequences coding for mutant Aequorea victoria green fluorescent protein GFP variants or proteins thereof requires a license from Amersham Biosciences under U S Patent Nos 5 625 048 5 777 079 6 054 321 and other pending U S and foreign patent applications In addition certain BD Biosciences Clontech products are made under U S Patent No 5 804 387 licensed from Stanford University Not For Profit research institutes or entities are granted an automatic license with the purchase of this product for use in non commercial internal research
7. B Stably Transfect and Select Double Stable Cell Lines 28 C Stably Transfect and Select Double Stable Cell Lines Cotransfection 30 BD Biosciences Clontech www bdbiosciences com Protocol PT3001 1 2 Version PR33678 Tet Systems User Manual Table of Contents continued D Screening Double Stable Cell Lines 31 E Working with Double Stable Cell Lines 31 X References 33 XI Related Products 35 Appendix A Vector Information 37 Appendix B Glossary 51 List of Figures Figure 1 Inducible on off control of gene expression in the Tet Systems 4 Figure 2 Schematic of gene regulation in the Tet Systems 6 Figure 3 Luciferase expression is rapidly induced in BD Tet Off cell line in response to removal of Dox 8 Figure 4 Developing Tet Off and Tet On Cell Lines 13 Figure 5 Fold induction of luciferase activity in different lots of FBS 21 Figure 6 Dose response curves for the CHO AA8 Luc Control Cell Line 23 Figure 7 Flow chart Developing Tet Cell Lines 26 Figure 8 Flow chart Developing double stable Tet Cell Lines 29 Figure 9 pTet Off and pTet On composite vector map 42 Figure 10 pTRE2hyg and pTRE2pur plasmid map and MCS 43 Figure 11 pTRE Tight vector map and MCS 44 Figure 12 pTRE Myc HA and 6xHN composite vector map and MCS 45 Figure 13 pTRE2Marker Myc HA and 6xHN composite vector map and MCS 46 Figure 14 pTK Hyg plasmid map 47 Figure 15 pTRE d2EGFP plasmid map 47 Figure 16 The expression cassette 48 Figur
8. TRE Figure 2 In the Tet Off System tTA binds the TRE and activates transcription in the absenceof Tc or Dox In the Tet On System rtTA binds the TRE and activates transcrip tion in the presence of Dox In both Tet On and Tet Off Systems transcrip tion is turned on or off in response to Dox in a precise and dose dependent manner You can greatly reduce the time needed to establish a Tet cell line by purchasing one of our premade Tet Cell Lines which already stably express the appropriate regulatory protein A list of available BD Tet Off and Tet On Cell Lines is available from our Tet Systems product page at www bdbiosciences com clontech Note that addition of a nuclear localization sequence n s to tTA or rtTA alters the protein s regulatory function M Gossen amp H Bujard pers comm Addition of an nis to tTA or rtTA increases maximum expression but also increases background expression due to altered binding affinity to tetO sequences unpublished observations Therefore we recommend that you do not add a nis to either tTA or rtTA for creating stable Tet cell lines C Advantages of the Tet Systems The BD Tet Off and Tet On systems have several advantages over other regulated gene expression systems that function in mammalian cells Extremely tight on off regulation Background or leaky expression of Gene X in the absence of induction is extremely low with pTRE or its variants Figure 1 Forthe lowest background e
9. TRE and actively silences transcription of Gene X Figure 18 As Dox is added to the culture medium the tTS dissociates from the TRE relieving transcriptional suppression At sufficient concentrations of Dox the rtTA transactivator binds the TRE and activates transcription of Gene X For additional information on pTet tTS including a vector map please referto the plTet tTS Vector Information Packet PT3334 5 available at www bdbiosciences com clontech 10 4 Induced high transcriptio Arbitrary light units 0 1 10 100 1 000 10 000 Doxycycline ng ml Figure 18 Dose response curve demonstrating controlled expression in a cell line co expressing tTS and rtTA HR5 cells which constitutively express rtTA were transiently trans fected with a plasmid expressing tTS and a control vector expressing luciferase downstream of the TRE Cells were cultured in the indicated levels of Dox After 24 hr cells were harvested and assayed for luciferase activity SD silencing domain AD activation domain Data provided courtesy of S Freundlieb Zentrum f r Molekulare Biologie ZMBH Universit t Heidelberg BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 50 Version PR33678 Tet Systems User Manual Appendix B Glossary Dox Double stable Tet Cell Line Gene X P CMV P minCMV hCMV 1 P tight Regulator Plasmid Response Plasmid rTetR
10. The next day examine the cells under a microscope If the cells were not rinsed upon thawing step 5 centrifuge cells if suspension cultures aspirate the medium and replace with fresh prewarmed complete medium without antibiotics Expand the culture as needed Note The appropriate selective antibiotic s may be added to the medium after 48 72 hr in culture D Preparing Frozen Stocks of Tet Cell Lines Once you have started growing a Tet Off or Tet On Cell Line from BD Biosciences Clontech prepare frozen aliquots to ensure a renewable source of cells Similarly prepare frozen aliquots of any double stable BD Tet Off or Tet On cell line or of any Tet Off or Tet On cell line that you make 1 3 Resuspend the pellet at a density of at least 1 2 x10 cells ml in Trypsinize the desired number of flasks 2 Pool cell suspensions together count cells and calculate total viable cell number Centrifuge cells at 125 x g for 10 min Aspirate the supernatant freezing medium Freezing medium can be purchased from Sigma C6164 or freeze cells in 70 9096 FBS 0 20 medium no addi tives and 1096 DMSO Dispense 1 ml aliquots into sterile cryovials Freeze slowly 1 C per min Nalgene makes cryo containers Nalgene 5100 for this purpose if a specialized freezer is not available freeze at 80 C overnight Alternatively place vials in athick walled styrofoam container at 20 C for 1 2 hr Tr
11. derived plasmids upon cointegration into the genome The sequence of pTK Hyg has been deposited in GenBank Accession 040398 Xhol 2 Scal Sac Il 443 3482 P minCMV r P emva d2EGFP pTRE d2EGFP Amp 4 0 kb EcoR 1292 1 1313 Xba 11319 1325 Hind III 1783 Figure 15 pTRE d2EGFP plasmid map p TRE d2EGFP is a response plasmid that can be used with the Tet Systems Clontechniques April 1998 d2EGFP a destabilized variant of the original EGFP is inserted between the Sac Il and EcoR sites in the pTRE MCS d2EGFP contains residues 422 461 of mouse ornithine decarboxylase MODO fused to the C terminus of EGFP This region of MODC contains a PEST amino acid sequence that targets the protein for degradation and results in rapid protein turnover d2EGFP has a half life of 2 hours as measured by fluorescence intensity of cells treated with the protein synthesis inhibitor cycloheximide Li X et al 1998 Protocol 4 PT3001 1 www bdbiosciences com BD Biosciences Clontech Version PR33678 47 Tet Systems User Manual Appendix A Vector Information continued Bidirectional Tet Vectors The Bidirectional Tet Vectors are used to simultaneously express two genes under control of a single TRE Baron et al 1995 for more information see Clontechniques October 1996 p 8 After a Tet Off or Tet On cell line is established a pBl vector is cotransfected with pTK Hyg to per
12. its com ponents to third parties Any other use of this product will require a license from BD Biosciences Clontech Please contact the Product Manager for Cell Biology by phone at either 800 662 2566 or 650 424 8222 extension 7816 or by e mail at licensing clontech com For Profit entities that wish to use this product in non commercial or commercial applications are required to obtain a license from BD Biosciences Clontech For license information please contact the Product Manager for Cell Biology by phone at either 800 662 2566 or 650 424 8222 extension 7816 or by e mail at licensing clontech com NucleoBond and NucleoSpin are registered trademarks of Macherey Nagel GmbH amp Co is a registered trademark of Molecular Dynamics Corporation BD BD logo and all other trademarks are the property of Becton Dickinson and Company 2003 BD Protocol PT3001 1 www bdbiosciences com BD Biosciences Clontech Version PR33678 55
13. method Transfect pT RE Gene X and a Linear Selection Marker pTK Hyg or pPUR in a ratio of between 10 1 and 20 1 by the desired method You may want to optimize ratios Note If desired the plasmids can be linearized by digestion with a restriction enzyme check the Vector Information Packets provided with each vector for appropriate restriction sites Plate transfected cells in ten 10 cm culture dishes each containing 10 ml of the appropriate complete medium at the optimal density determined in Section VII Allow cells to divide twice 24 48 hr time may vary with cell line then add hygromycin or puromycin to 200 400 ug ml or the optimal concentration determined in Section VII BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 30 Version PR33678 Tet Systems User Manual IX Development of Double Stable Cell Lines continued For Tet Off cells only When establishing a double stable Tet Off cell line you may wish to culture the cells in the presence of 2 ug ml Tc or 1 ug ml Dox in order to keep transcription of Gene X turned off This is essential if Protein X is toxic to the cell 5 Replace medium with fresh complete medium containing hygromycin or puromycin every four days Fresh Dox MUST be added every two days for Tet Off cells After about five days cells should start to die Split cells if they reach confluency before massive cell death begins After 2 4 weeks hyg or
14. or as soon thereafter as possible Increased loss of viability may occur after shipping if culturing is delayed 1 Thaw vial of cells rapidly in a 37 water bath with constant agitation Immediately upon thawing wipe the outside of the vial with 70 EtOH Transfer the contents of the vial to a 10 cm dish ora T25 or T75 flask containing 1 ml of medium without antibiotics Mix gently 2 Add an additional 4 ml of medium to the flask dish and mix gently 3 Add additional medium to the culture as follows T25 flask or 10 cm dish add 5 ml T75 flask add 10 ml Note for Jurkat and other suspension cultures suspend cells at a density of no less than 2x105 cells ml in the appropriate medium Protocol 4 PT3001 1 www bdbiosciences com BD Biosciences Clontech Version PR33678 19 Tet Systems User Manual VI Cell Culture Guidelines continued 4 Mix the cell suspension thoroughly Gently rock or swirl the dish flask to distribute the cells evenly over the growth surface and place it in a 37 C humidified incubator 5 1096 CO as appropriate Alternative method The cells can also be rinsed prior to incubation If rinsing is desired perform steps 1 and 2 in a 15 ml conical centrifuge tube Centrifuge at 125 x g for 10 min and resuspend in complete medium for culturing This step removes the cryopreservative and can be beneficial when resuspending in small volumes However this step can damage fragile cell membranes
15. puro resistant colonies will begin to appear 6 Using cloning cylinders or discs isolate large healthy colonies and transfer them to individual plates or wells Isolate as many clones as possible D Screening Double Stable Cell Lines 1 Test isolated resistant clones for Dox regulated gene expression by dividing a suitable number of cells in half and testing for Gene X expression or pBl reporter expression in the presence and absence of 1 ug ml Dox As with the development of Tet Off or Tet On cell lines you should generally choose the cell line that gives you the highest overall induction and lowest background i e uninduced expression level of Gene X 2 Allow the cells to grow for at least 48 hr then assay each sample for Gene X expression using one of the methods described in Section A 3 Optional Confirm the presence of integrated pTRE Gene X by per forming PCR on chromosomal DNA using primers that will amplify an internal portion of the plasmid 4 Once you have developed a suitable double stable Tet Off or Tet On cell line prepare frozen aliquots to ensure a renewable source of the cells Section VI D E Working with Double Stable Tet Cell Lines The Tet System has been established successfully in many cell types as well as transgenic mice rats plants and yeast In general failure to obtain a cell line with a low background level of Gene X expression is a result of the integration site in the tested li
16. purposes the terms of which are disclosed in detail in the license that accompanies the shipment of this product Such license specifically ex cludes the right to sell or otherwise transfer this product or its components to third parties For Profit research institutes or entities must obtain a license from Amersham Biosciences E mail gfp amershambiosciences com Please contact BD Biosciences Clontech directly for any other assistance including purchasing and technical support All companies and institutions purchasing Living Colors products will be in cluded in a quarterly report to Aurora Biosciences as required by the BD Biosciences Clontech Aurora Biosciences license agreement BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 54 Version PR33678 Tet Systems User Manual Notice to Purchaser continued BD Biosciences Clontech s red fluorescent protein DsRed and its variants are the subject of pend ing U S and foreign patents Not For Profit Entities Orders may be placed in the normal manner by contacting your local repre sentative or BD Biosciences Clontech Customer Service at either 800 662 2566 or 650 424 8222 extension 1 BD Biosciences Clontech grants not for profit research entities a worldwide non exclu sive royalty free limited license to use this product for non commercial life science research use only Such license specifically excludes the right to sell or otherwise transfer this product or
17. the following general references Culture of Animal Cells Fourth Edition ed by R I Freshney 2000 Wiley Liss NY e Current Protocols in Molecular Biology ed by F M Ausubel et al 1995 Wiley amp Sons B Characteristics of BD Tet Off and Tet On Cell Lines See the Product Analysis Certificate PAC for information on each Tet Off and Tet On Cell Line Additional information for all the currently available Tet Off and Tet On Cell Lines including propagation information is provided in documents PT3001 2 and PT3001 3 available from our Tet Systems product page at www bdbiosciences com clontech General cell culture conditions Premade Tet Off and Tet On Cell Lines should be grown at 37 C in a humidified chamber with 5 10 CO See the PAC for details particular to each cell line Relative growth rates The incubation times in this User Manual are for cells such as CHO or HeLa with relatively rapid doubling times Other cell types will differ in their growth rates Selection in G418 and hygromycin Maintain stable and double stable Tet Off and Tet On Cell Lines in the appropriate selective medium how ever the concentration can be reduced typically to 100 ug ml for each drug from the levels used to select stably transfected clones You may wish to alternate between selecting and nonselecting conditions C Starting Tet Cell Cultures From Frozen Stocks Note Frozen cells should be cultured immediately upon receipt
18. 01 3 BD Tet Off and BD Tet On Cell Lines 1 0 ml Tet Off or Tet On Cell Line 2 x 108 cells ml 0 5 ml CHO AA8 Luc Tet Off Control Cell Line 1 x 10 cells 50 ml Tet System Approved Fetal Bovine Serum e Tet Cell Lines Protocol at a Glance PT3001 2 List of Available Tet Cell Lines PT3001 3 BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 14 Version PR33678 Tet Systems User Manual IV Additional Materials Required For cell culture Dulbecco s Modified Eagle s Medium DMEM BD 234627 Alpha Minimal Essential Medium Eagle alpha MEM RPMI 1640 or other specified medium The appropriate medium for growing BD Biosciences Clontech s premade Tet Off and Tet On cell lines is described on the Product Analysis Certificate provided with each cell line Fetal bovine serum FBS It is critical that the FBS not inhibit Tet responsive expression You can eliminate Tc contamination problems by using BD Biosciences Clontech s Tet System Approved FBS US Sourced 631101 and USDA Approved 631106 This serum has been functionally tested in the Tet Systems 10 ensure against possible Tc contamination Alternatively use the CHO AA8 Luc Control Cell Line to test for Tc contamination in other sera as described in Section VIIA Note The PC 12 Tet Off and Tet On Cell Lines require horse serum Sigma 0146 for growth which does not normally contain Tc 200 mM L Glutamine Sigma G7513 Solution o
19. 10 1 x 103 1 x 107 0 1 1 0 and 10 0 ug ml To titrate Dox add Dox to final concentrations of 0 1 x 10 1 x 107 0 1 1 0 10 and 100 ng ml 3 Allow the cells to grow for 48 hr 4 Assay each sample for luciferase activity using any standard luciferase assay Plot your results logarithmically and compare to Figure 6 B Titrating G418 Hygromycin and Puromycin Kill Curves Prior to using G418 hygromycin or puromycin to establish stable and double stable cell lines it is important titrate your selection agent stocks to determine the optimal concentration for selection with the particular host cell line being tested This is also important because of lot to lot variation in the potency of these drugs Therefore you should titrate each new lot of antibiotic to determine the optimal concentration We recommend that you perform two experiments for each drug 1 a titration to determine the optimal drug concentration and 2 an experimentto determine the optimal plating density This step is recommended even if you are using premade Tet Cell Lines 1 Titrate at fixed cell density a Plate 2 x 10 cells in each of six 10 cm tissue culture dishes containing 10 ml of the appropriate complete medium plus varying amounts 0 50 100 200 400 800 ug ml of hygromycin or G418 For puromycin add the drug at 0 1 2 5 5 7 5 and 10 ug ml Note 293 Tet On Tet Off cells 630903 and 630908 are especially sensitive to h
20. 493 I8d 6 c8 9 euou 4499 7 4493 189 9 0 80 ers euou 493 4493 189 ez 1494 pue 5 1gd S E L E 29 96 euou jeb g L lad 9661 12 19 uoreg 719 1ad X 6 80 9 euou eseJeyon 2189 9661 72 1e 7 99 9 0 X G E L E eqx 4 euou jeb g 1gd 9661 72 19 uoieg 9 gd A LO LE qx 9 v euou X utejoud 194 9661 12 19 194 s eu Azue 992u949J91 59215 00112134591 215 e qeioojes 55 OWEN juowbei4 onsoubeiq NOILVINHOJNI HOLO3A SINALSAS LAL Ill 318V L BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 Version PR33678 41 Tet Systems User Manual Appendix A Vector Information continued An annotated printout of the pTRE2hyg sequence PT3521 5 is provided with the Tet Off and Tet On Gene Expression Systems You can obtain the sequences of the other vectors at www bdbiosciences com clontech Xho 2 Hind pTet On only 871 Mutations that convert TetR to rTetR d tTA to cal Bsal tan 3949 3546 Figure 9 and pTet On composite vector map Unique sites are in bold Only pTet On contains the second Hind Ill
21. Anhydrotetracycline Other Related Products BD Biosciences Clontech 36 NucleoBond and NucleoSpin Columns NucleoTrap Gel Extraction kit NucleoTrap PCR Purification Kit Luciferase Reporter Assay Kit BD Creator pDNR Cloning Kits pCMVB Vector pEGFP N1 Vector www bdbiosciences com Cat New Cat K1626 1 631020 K1627 1 631021 K1674 1 631023 K1675 1 631024 6140 1 631003 6159 1 631007 6134 1 631001 6137 1 631002 6347 1 631015 many many K1651 1 631022 K1652 1 631050 8484 1 631058 8483 1 631057 8630 1 631101 8637 1 631106 K2051 1 631312 8020 1 631301 8056 1 631307 8057 1 631309 8634 1 631311 8052 1 631305 8633 1 631310 many many K3070 1 636018 K3071 1 636020 K2039 1 631714 K1670 1 631615 6177 1 631719 6085 1 632318 Protocol PT3001 1 Version PR33678 Appendix A Vector Information Tet Systems User Manual Table BD Tet Off and Tet On Vector alignment Applications Basic Vectors pTet Off Pew tetR VP16 Regulator vector for use in Tet Off system pTet 0n Pew 16 Regulator vector for use in Tet On system pTRE2 TRE gene of interest poly A Response plasmids encoding the Tet Responsive Element TRE pTRE2
22. BD Tet Off and Tet On Gene Expression Systems User Manual Cat No 630921 or K1620 1 630922 or K1621 1 PT3001 1 PR33678 Published 03 14 2003 Tet Systems User Manual Table of Contents VI VII Vill Introduction 4 A Summary 4 B The BD Tet Off and Tet On Systems 5 C Advantages of the Tet Systems 7 D Tet Off vs Tet On Systems 9 E Tetracycline vs Doxycycline 9 F Additional Tet Response Vectors 10 G Beyond the Basics pBl VP16 and pTet tTS Vectors 11 H Retroviral Tet Expression 11 l Adenoviral Tet Expression 11 Protocol Overview 12 List of Components 14 Additional Materials Required 15 Plasmid Manipulations 18 A Propagation of Vector Plasmids 18 B Generating your Gene Specific Expression Vector 18 Cell Culture Guidelines 19 A General Information 19 B Characteristics of Tet Off and Tet On Cell Lines 19 C Starting Tet Cell Cultures from Frozen Stocks 19 D Preparing Frozen Stocks of Tet Cell Lines 20 Pilot Experiments 21 A Pilot Experiment with the CHO AA8 Luc Tet Off Control Cell Line 21 B Titrating G418 Hygromycin and Puromycin Kill Curves 22 C Test Potential Host Cells by Transient Transfection with pTRE2hyg Luc and pTet Off or pTet On 24 Development of Stable Cell Lines 25 A Transfection and Selection of Stable Cell Lines 25 B Screening Stable Cell Lines 27 Development of Double Stable Cell Lines 28 A Test pTRE Gene X by Transient Transfection into a Tet Off or Tet On Cell Line 28
23. FP contains the gene encoding EGFP cloned into the and Hind III sites in the pTRE Tight MCS EGFP is an enhanced variant of the Aequorea victoria green fluorescent protein pTRE Tight DsRed2 contains the gene encoding DsRed2 cloned into the BamH and Not sites in the pTRE Tight MCS DsRed2 is a variant of the red fluorescent protein isolated from the IndoPacific sea anemone relative Discosoma sp BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 44 Version PR33678 Tet Systems User Manual Appendix A Vector Information continued MCS hCMV 1 pTRE Myc HA amp 6xHN 3 8 kb p uU P tag B globin polyA bd c Myc HA or 6xHN epitope tag ATG XXX XXX XXX XXX XXX XXX XXX XXX XXX XXX XXX XXX XXX CTT ATG GCC ATG GAG GCC Sfil CAA GCT TGG TCG ACC GAG ATC TCT CGA GGT ACC GCG GCC GCT CGA CGA TAT CTC TAG A Hind 111 Sall Noti EcoRV Xbal Figure 12 pTRE Myc HA and 6xHN composite vector map and multiple clone site MCS These tagged pTRE vectors contain an MCS immediately downstream of the Tet responsive 1 promoter Pyoyys 1 contains the Tet response element TRE which consists of seven copies of a sequence containing the 19 bp tet operator sequence ftetO and the minimal CMV promoter which lacks the enhancer that is part of the complete CMV promoter in the regulatory plasmids Consequently Pacmv 1 is silent in the absence of bi
24. GCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTCTAGA Nhel Eagl Clal Hindili Sall EcoRV Xbal Figure 11 pTRE Tight vector map MCS This response plasmid contains an MCS immedi ately downstream of the Tet responsive promoter Py contains a modified Tet response element TRE which consists of seven direct repeats ol a 36 bp sequence that contains the 19 bp tet operator sequence fetO and the minimal CMV promoter Pmincmva Which lacks the enhancer that is part of the complete CMV promoter Consequently Pign is silent in the absence of binding of TetR or rTetR to the tetO sequences Genes inserted into the MCS will be responsive to the tTA and rtTA regulatory proteins in the Tet Off and Tet On systems respectively Note that the cloned insert must have an initiating ATG codon The addition of a Kozak sequence is not required but may improve expression levels pTRE Tight Gene X plasmids should be cotransfected with the Linear Hygromycin Marker 631625 not included or Linear Puromycin Marker 631626 not included to permit selection of stable transfectants Complete sequence information is provided in the pTRE Tight Vector Information Packet PT3720 5 The pTRE Tight Luc Control Vector packaged with the pTRE Tight Vector contains an additional 1 649 bp encoding firefly luciferase inserted into the MCS This vector can be used as a reporter of induction efficiency It is not intended as a cloning vector pTRE Tight EG
25. Luc cell line open circles The difference in background and induction levels between CHO AA8 Luc and its sibling cell line demonstrates the importance of screening multiple independent clonal lines when establishing double stable Tet Off Cell Lines see Section IX C Panel B Results of a separate experiment comparing Tc open circles and Dox closed circles dose response curves for the CHO AA8 Luc Control Cell Line Experiments with another control cell CHO K1 EGFP Luc Tet Off have demonstrated that suppression can be maintained with Dox concentrations as low as 10 pg ml Cunningham et al 1997 3 Once you have determined the optimal drug concentration determine the optimal plating density by plating cells at several different densities in the presence of a constant amount of drug If cells are plated at too high a density they will reach confluency before the selection takes effect Optimal plating density is dependent on population doubling time and cell surface area For example large cells that double rapidly have a lower optimal plating density than small cells that double slowly a Plate cells at several different densities in each of six 10 cm tissue culture dishes containing 10 ml of the appropriate selective medium Suggested densities cells 10 cm dish 5 x 106 1 x 106 5 x 105 2 x 105 1 x 105 and 5 x 10 b Incubate the cells for 5 14 days replacing the selective medium every four days c Examine the di
26. Tet Off Tet Off System eS binds TRE and activates transcription iM PN tetR VP16 in the absence of Dox REMOVE DOX Transcription e TRE Gene of interest Le UU TRE gu Gene of interest BD Tet On System rtTA 6 binds TRE and activates transcription EMT m wri in the presence of Dox S BOE _ DOX Tran3 lt tiption Transcription Gene of interest Gene of interest ADD DOX o Figure 2 Schematic of gene regulation in the BD Tet Off and Tet On Systems Tet Off The TRE is located upstream of the minimal immediate early promoter of cytomegalovirus which is silent in the absence of activation tTA binds the TRE and thereby activates transcription of Gene X in the absence of Tc or Dox Tet On The reverse Tet repressor rTetR was created by four amino acid changes that reverse the protein s response to Dox As a result of these changes the rTetR domain of rtTA binds the TRE and activates transcription in the presence of Dox Please see Appendix A for maps and detailed vector information BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 6 Version PR33678 Tet Systems User Manual Introduction continued the response e g pTRE Gene X Vectors Gene X is only expressed upon binding of the tTA or rtTA protein to the
27. alactosidase or EGFP BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 48 Version PR33678 Tet Systems User Manual Appendix A Vector Information continued VP16 Minimal Domain Vectors The VP16 Minimal Domain Vector Set 631019 ptTA2 ptTA3 and ptTA4 expresses tetracycline controlled transactivators containing modified VP16 ac tivation domains Figure 17 Baron et al 1997 Overexpression of unmodified VP16 can have negative pleiotropic effects due to interactions with essential components of the transcriptional machinery This generally does not interfere with in vitro expression but can pose problems in vivo when transcription is driven by a strong tissue specific promoter The modified VP16 moieties contained in these transactivators allows their expression at higher intracellular levels potentially allowing increased stability for cell culture and transgenic applications Baron et al 1997 Furthermore each vector allows protein expression over a different induction range Panel B Applications such as knock in knock out experiments rely on site specific inte gration and thus are dependent on the transcriptional activity of the particular locus In these situations the VP16 Minimal Domain Vectors may enable you to obtain optimal expression levels by adapting the activation potential of the transactivator to the expression level of the locus tTA VP16 minimal Neo
28. an be used 5 or 3 to sequence junctions between an insert and any of the Tet system response vectors They can also be used to amplify or confirm the presence of inserts via PCR when the expected insert size is less than 2 3 kb See Appendix A for additional information on these vectors BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 10 Version PR33678 Tet Systems User Manual Introduction continued G Beyond the Basics pBl VP16 and pTet tTS Vectors The Bidirectional pBl Tet Vectors are specially designed response vec tors that allow coregulated expression of two genes under control of a single TRE They are ideal response vectors to use if you do not have a functional assay for your gene of interest because you can select for expression of the coregulated marker gene either B galactosidase luciferase or EGFP These vectors do not contain a selectable gene and should be cotransfected with one of the Linear Selection Markers 631625 or 44631626 pTK Hyg or pPUR The VP16 Minimal Domain Vectors 631019 contain a protein fusion of TetR fused to altered VP16 activation domains These proteins are toler ated at higher intracellular levels and have a better transfection efficiency in some cell types They are especially useful in transgenic studies where high expression of unmodified VP16 may be toxic to cells ThepTet tTS Vector 631011 is designedto prevent unregulated leaky gene express
29. ansfer to 80 C overnight Remove vials from styrofoam container or cryo containers the following day and place in liquid nitrogen storage or ultra low temperature freezer 150 C Two or more weeks later Plate a vial of frozen cells as described in Section C to confirm viability BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 20 Version PR33678 Tet Systems User Manual VII Pilot Experiments A Pilot Experiment with the CHO AA8 Luc Tet Off Control Cell Line Before you perform any other experiments we strongly recommend that you perform a dose response curve with the CHO AA8 Luc Tet Off Control Cell Line This is a premade double stable Tet Off Cell Line that exhibits over 10 fold induction of luciferase upon removal of Tc or Dox from the culture medium Figure 5 In addition to providing a hands on introduc tion to the Tet Systems this experiment serves two critical functions Determination of effective concentrations of Tc or Dox stocks The concentrations of Tc and Dox listed throughout this protocol are approximate The optimal concentration may vary with different cell lines and with different lots of antibiotic In general full repression of gene expression in Tet Off lines can be obtained with 1 2 ug ml Tc or 10 ng 1 ug ml Dox Full activation of gene expression in Tet On cell lines can be obtained with 100 ng 1 ug ml Dox Testing of serum for Tc contamination As shown in Figu
30. ciences Clontech Version PR33678 25 Tet Systems User Manual VIII Development of Cell Lines continued Neo NA Neo E OR Transfect host cell line with regulator plasmid pTet Off or pTet On Select in presence of G418 Y solate at least 30 G418 resistant clones e du ae e Screen by transient transfections with pTRE2hyg Luc for clones with low background and high induction of luciferase in response to Tc or Dox Freeze stocks of Tet cell line 7 Tet Off or Tet On cell line Figure 7 Flow chart for developing Tet cell lines BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 Version PR33678 Tet Systems User Manual VIII Development of Cell Lines continued B Screening Stable Cell Lines The next step is to perform transient transfection assays with pTRE2hyg Luc or another reporter vector such as pBI EGFP Luc or pTRE d2EGFP to identify G418 resistant clones that meet the criteria for stable Tet Off or Tet On cell lines See Appendix for maps and more information on these reporter vectors 1 2 Pick clones and expand as needed for your particular line Screen clones once they reach 50 80 confluency in a 6 well plate Trypsinizethe cells and split about 1 3 into a single well of a 6 well plate The cells in this stock plate will be propagated depending upon the results of the screening assay Transfect the remaining 2 3 of the cells w
31. control of the tetracycline response element or TRE We provide two response vector series for the Tet Systems Our original vector series pTRE or its variants contain the TRE which consists of seven direct repeats of a 42 bp sequence contain ing the tetO located just upstream of the minimal CMV promoter Prisci Pmincmy lacks the strong enhancer elements normally associated with the CMV immediate early promoter Because these enhancer elements are missing there is extremely low background expression of Gene X from the TRE in the absence of binding by the TetR domain of tTA or the rTetR domain of rtTA Our second response vector series pTRE Tight contain a modified TRE TREmoa upstream of an altered minimal CMV promoter Princmva s resulting in further reduced basal expression of Gene X pTRE Tight can fully minimize background expression in certain cell lines and is especially useful in cases where background expression is unacceptable such as the expression of proteins that are extremely potent or toxic to the host cell April 2003 Clontechniques The ultimate goal in setting up a functional Tet System is creating double stable Tet cell line which contains both the regulatory and response plasmids When cells contain both the regulatory pTet Off or pTet On and Protocol 4 PT3001 1 www bdbiosciences com BD Biosciences Clontech Version PR33678 5 Tet Systems User Manual Introduction continued B D M
32. d 9UN Qu611 3u 1d ym 91992 9 Hueg ev euou ese1gjion 1 1 1 1009A 90 80 oux 9 z euou X ulajoud 1uBi1 3u1d 914 0 21 02 18097 90vS euou 7 23419 10109 LO0 0 4024 gle euou 23819 vel Gr IHueg pepiaoid GGL Y S oux 9 eseJejion onTandz3u 1d Lg Hweg 10198A GGL ys oux Lg X urejoud ndz3u1d BAuz3t Ld GGL ys oux 0969 on1 6Auz3u 1d Hweg J0199A GGL GLE 104X 6 upuojD u X J0199A L 0E 814097 ev auou su 1 9 SHNd 6661 72 19 qeipunej4 60 SL II 109A 6 8104X 282 Uulo uoeu VLU oeu LaHnd 9661 72 19 uessot uO e d EZ IIl 2661 uessoo 10199A 6 8104X 464 YL o ul GLAHNd 661 72 Ja xzyused s eu Azue 92u949J91 92u9819JoH 59215 0112114591 9216 juowbei4 onsouDeiq NOILVINYOANI OLOJA SWALSAS LAL 319 1 BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 Version PR33678 39 Tet Systems User Manual d ion continue Vector Informati Appendix A
33. d induction in transient expression assays but stable clones can be isolated that exhibit 6 000 fold induction and background expression levels that are indistin guishable from control background expression Therefore an apparent lack of induction response in the transient assay should not be the sole reason for aborting your experiments in a particular cell line BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 24 Version PR33678 Tet Systems User Manual VIII Development of Stable Tet Cell Lines A SKIP SECTION IF YOU HAVE PURCHASED PREMADE BD BD Tet On CELL LINE Transfection and Selection of Stable Cell Lines Figure 7 The following protocol describes the development of Tet Off or Tet On cell lines You must optimize the protocol for each cell type Some of the parameters most likely to need adjustment are plating densities transfec tion method G418 concentrations for selection and incubation and grow ing times Regardless of the cell type and transfection method the goal is to generate acellline that gives low background and high induction of luciferase activity when tested by transient transfection with pTRE2hyg Luc in Section B Because the level of expression of tTA or rtTA is profoundly affected by the site of integration we recommend that you isolate and analyze as many clones as possible at Step 6 In general test at least 30 clones We have screened as man
34. d method such as the NucleoTrap Gel Extraction Kit 636018 or NucleoTrap PCR Purifica tion Kit 636020 The cDNA or gene fragment must contain an ATG initiation codon In some cases addition of a Kozak consensus ribo some binding site Kozak 1987 may improve expression levels however many genes have been efficiently expressed in Tet systems without the addition of a Kozak sequence The fragment can be generated using compatible restriction sites that are present on either side of the gene and in the cloning vector If no such sites are present the gene fragment can be generated by PCR with suitable restriction sites incorporated into the primers 2 Digest the response vector pTRE or its variant with the appropriate restriction enzyme s treat with phosphatase and 3 Ligate the response vector and the Gene X fragment Transform ligation mixtures into E coli 5 Identify the desired recombinant plasmid by restriction analysis and confirm orientation and junctions by sequencing A BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 18 Version PR33678 Tet Systems User Manual VI Cell Culture Guidelines A General Information The protocols in this User Manual provide only general guidelines for mammalian cell culture techniques Perform all steps involving cell culture using sterile technique in a suitable hood For those requiring more information on mammalian cell culture we recommend
35. e 17 VP16 Minimal Domain vectors 49 Figure 18 Controlled expression in a cell line co expressing tTS and rtTA 50 List of Tables Tablel Tet Off and Tet On Vector Alignment 37 Table Tet Systems Vector Information 39 Version 4 PR33678 3 Tet Systems User Manual 1 Introduction A Summary The BD Tet Off and BD Tet On Gene Expression Systems and the premade BD Tet Off and Tet On Cell Lines give researchers ready access to the regulated high level gene expression systems de scribed by Gossen amp Bujard 1992 Tet Off and Gossen et al 1995 Tet On In the Tet Off system gene expression is turned on when tetracycline Tc or doxycycline Dox a Tc derivative is removed from the culture medium In contrast expression is turned on in the Tet On system by the addition of Dox Figure 1A The Tet On system is responsive only to Dox not to Tc Both systems permit gene expression to be tightly regulated in response to varying concentrations of Tc or Dox Figure 1B Maximal expression levels in Tet systems are very high and compare favorably with the maximal levels obtainable from strong constitutive mammalian promoters such as CMV Yin et al 1996 Unlike other inducible mammalian expression systems gene regulation in the Tet Systems is highly specific so interpretation of results is not complicated by pleiotropic effects or nonspecific induction Tc BD Tet Off BD Tet On ng ml 2 000 6 4 2 1 05 025 0 Do
36. e inducer in Tet systems Gossen amp Bujard 1993 Affinity for TetR and antibiotic potency are apparently mediated by different chemical moieties some derivatives such as anhydrotetracycline have an increased affinity for TetR and decreased antibiotic activity Gossen et al 1993 Protocol 4 PT3001 1 www bdbiosciences com BD Biosciences Clontech Version PR33678 9 Tet Systems User Manual Introduction continued F Additional Tet Response Vectors The complete BD Tet Off and Tet On Gene Expression Systems are provided with pTRE2hyg as the response vector In addition to the TRE regulatory element and a multiple cloning site this vector also expresses the hygromycin resistance gene permitting easy selection of stable trans fectants We also offer pTRE2pur 631013 for an alternative selection scheme using puromycin Another response vector the pTRE Tight Vector 631059 is available separately pTRE Tight contains a modified TRE element TRE moa that can minimize basal expression in certain cell lines This vector is also offered in two reporter formats The pTRE Tight EGFP Vector features the gene for enhanced green fluorescent protein cloned into the pTRE Tight Vector while the pTRE Tight DsRed2 Vector expresses a variant of our original red fluorescent protein These vectors do not contain a selectable gene and for best results should be cotransfected with our Linear Selection Marker for hygromycin 631625 or purom
37. e of Tc Reverse tetracycline controlled transactivator A 37 kDa fusion pro tein consisting of the rTetR and the VP16 activation domain AD Binds specifically to TRE and activates transcription in the presence of Dox The chemical compound tetracycline Tetracycline asinthe tetoperonorthe Tetrepressor Thecompound tetracycline is abbreviated Tc Any cell line that stably expresses tTA from integrated copies of pTet Off Tet Off cell lines can either be made by the researcher or purchased from BD Biosciences Clontech www bdbiosciences com BD Biosciences Clontech Tet Systems User Manual Appendix B Glossary continued BD Tet On Cell Lines letO TetR TRE TRE mod tTA tTS VP16 AD Any cell line that stably expresses rtTA from integrated copies of pTet On Tet On cell lines can either be made by the researcher or purchased from BD Biosciences Clontech The tet operator a 19 bp cis acting regulatory DNA sequence from the bacterial tet operon where it is the natural binding site for TetR See TRE The Tet repressor component of tTA and rtTA In E coli TetR binds specifically to tetO and blocks transcription of the tet operon in the absence of Tc Tet Response Element A regulatory sequence consisting of seven direct repeats of a 42 bp sequence that contains the tetO Modified Tet Response Element A regulatory sequence consisting of seven direct repeats of a 36 bp sequence that contains the f
38. ecommend that you culture the cells in the presence of 2ug ml Tc or 1 ug ml inorderto keep transcription of Gene X turned off This is essential if Protein X is toxic to the cell Replace medium with fresh complete medium containing the selection antibiotic hyg or pur every four days Fresh Dox MUST be added every two days for Tet Off cells After about five days cells should start to die Split cells if they reach confluency before massive cell death begins After 2 4 weeks hyg resistant or pur resistant colonies will begin to appear Isolate large healthy colonies and transfer them to individual plates or wells Isolate as many clones as possible Proceed to Section IX D C Stably Transfectand Select Double Stable Cell Lines Cotransfection pTRE2hyg Gene X and pTRE2pur Gene X response plasmids contain a selection marker in the backbone Other pTRE response plasmids which do not contain a marker must be cotransfected with a selection vector such as a Linear Selection Marker pTK Hyg or pPUR using the following protocol Note If you are using a selection vector other than a Linear Selection Marker pTK Hyg or pPUR the promoter should not contain an enhancer element If it does cointegration of the response and selection plasmids may lead to high background expression of Gene X in the uninduced state 1 2 Grow cells to 80 confluency in complete medium or to a density appropriate for your transfection
39. etO Tetracycline controlled transactivator A 37 kDa fusion protein con sisting of the TetR and the VP16 activation domain AD Binds specifically to the TRE and activates transcription in the absence of Tc or Dox Tetracycline controlled transcriptional silencer a fusion protein con sisting of the TetR and the KRAB AB domain of Kid 1 Binds specifically to the TRE and suppresses transcription in the absence of Dox The activation domain of the VP16 protein from herpes simplex virus BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 52 Version PR33678 Tet Systems User Manual Notes Protocol 4 PT3001 1 www bdbiosciences com BD Biosciences Clontech Version PR33678 53 Tet Systems User Manual Notice to Purchaser This product is intended to be used for research purposes only It is not to be used for drug or diagnostic purposes nor is it intended for human use BD Biosciences Clontech products may not be resold modified for resale or used to manufacture commercial products without the written approval of BD Biosciences Clontech Use of the Tetracycline controllable expression systems the Tet Technology is covered by a series of patents including U S patents 45 464 758 and 5 814 618 which are proprietary to Abbott Labo ratories Academic research institutions are granted an automatic license with the purchase of this product to use the Tet Technology only for internal academic research purposes
40. f 10 000 units ml Penicillin G sodium and 10 000 ug ml Streptomycin sulfate Sigma P0781 Antibiotics for clonal selection Prior to use determine the optimal concentration of each antibiotic for selection as described in Section VII B G418 for selection of Tet Off and Tet On Cell Lines G418 is available in powdered form from BD Biosciences Clontech 631307 Note that the effective weight is about 0 7 g per gram of powder Make a 10 mg ml stock solution by dissolving 1 g of powder in approxi mately 70 ml of DMEM or alpha MEM without supplements Filter sterilize and store at 4 C Recommended working concentration Maintenance 100 pg ml Selection HeLa or CHO cells 400 500 ug ml acceptable range 50 800 ug ml Hygromycin for selection of double stable Tet Off and Tet On Cell Lines Hygromycin B is available from BD Biosciences Clontech 631309 Recommended working concentration Maintenance 100 ug ml Selection HeLa or CHO cells 200 ug ml acceptable range 50 800 ug ml Puromycin for maintenance of the MDCK Tet Off Cell Line and for selection of double stable Tet On and Tet Off cells Available from BD Biosciences Clontech 631305 631306 Recommended working concentration Maintenance 0 5 ug ml Selection acceptable range 0 5 5 ug ml Protocol PT3001 1 www bdbiosciences com BD Biosciences Clontech Version PR33678 15 Tet Systems User Manual IV Additional Materials Required continued Tryp
41. h consists of seven copies of a sequence containing the 19 bp tet operator sequence 1210 and the minimal CMV promoter which lacks the enhancer that is part of the complete CMV promoter in the regulatory plasmids Consequently is silent in the absence of binding of TetR or rTetR to the tetO sequences Genes inserted into one of the sites in the MCS will be responsive to the tTA and rtTA regulatory proteins in the Tet Off and Tet On systems respectively Note that the cloned insert must have an initiating ATG codon The addition of a Kozak sequence is not required but may improve expression levels The addition of an internal selection element Hyg or eliminates the need for cotransfection with pTK Hyg Complete sequence information is provided in the pTRE2hyg and pTRE2pur Vector Information Packets pTRE2hyg PT3521 5 pTRE2pur PT3520 5 pTRE2hyg Luc and pTRE2pur Luc contain the gene encoding firefly luciferase cloned into the BamH 1 Nhel sites in the pTRE2hyg and pTRE2pur MCS The sites were destroyed during construction The luciferase construct adds 1 649 bp to the vectors Protocol 4 PT3001 1 www bdbiosciences com BD Biosciences Clontech Version PR33678 43 Tet Systems User Manual Appendix A Vector Information continued MCS 323 411 pTRE Tight 2 6 kh Xho 602 323 GAATTCGAGCTCGGTACCCGGGGATCCTCTAGTCAGCTGACGCGT EcoR BamH 1 Pvul Mlul 368 Smal
42. he Tet responsive 1 promoter 1 contains the Tet response element TRE which consists of seven copies of a sequence containing the 19 bp tet operator sequence tetO and the minimal CMV promoter which lacks the enhancer that is part of the complete CMV promoter in the regulatory plasmids Consequently 15 Silent in the absence of binding of tTA or rtTA to the tetO sequences Genes inserted into one of the sites in the MCS will be responsive to the tTA and rtTA regulatory proteins in the Tet Off and Tet On systems respectively The tagged fusion protein can be efficiently detected and purified using antibodies and resins optimized against the different markers Complete sequence informa tion is provided in the Vector Information Packets pTRE2hyg2 Myc PT3685 5 pTRE2hyg2 HA PT3684 5 pTRE2hyg2 6xHN PT3686 5 pTRE2pur Myc PT3688 5 pTRE2pur HA PT3687 5 pTRE2pur 6xHN PT3689 5 BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 46 Version PR33678 Tet Systems User Manual Appendix A Vector Information continued 5040 EcoR 1017 EcoR eng EcoR 2330 Figure 14 pTK Hyg plasmid map pTK Hyg is cotransfected with pT RE derived plasmids but not with pTRE2hyg and pTRE2pur vectors to allow selection of stably transformed cell lines in the presence of hygromycin The absence of an enhancer element on pTK Hyg prevents the unwanted activation of pTRE
43. hyg pur TRE Paw gene of interest poly H Hyg Pur for use in either Tet Off or Tet On pTRE Tight TRE Faam gene of interest Poly A Response plasmid encoding a modified Tet Responsive Element for use in either Tet Off or Tet On Accessory Vectors id For tighter control of gene expression in t 5 pTet tTS a Systems 5 Paw tetR 16 2 3 4 _ Minimal domain vectors used in ptTA 2 3 4 Tet Off System minimizes VP16 toxicity i i Reporter or control vector in either TRE d2EGFP TRE Pmincmv gene of interest polyA p E Tet Off or Tet On pTRE Tight EGFP TRE poly A Reporter vector containing a modified Tet Responsive Element TRE moa pTRE Tight DsRed2 _ Ho D polya for use in either Tet Off or Tet On Tagged Vectors _ 2 fi poly AL Response plasmids for use in either P M3 Tet Off or Tet On System pTRE HA re Pew of interest poly Used for screening with antibodies or for purification pTRE 6xHN re 6xHN gene of interest poly A Bidirectional Tet Vectors pBI EGFP EGFP PminCMV TRE PmincMV gene of interest pBI G gene of intorost rc TRE PmincMv lacZ Response vectors for monitoring expression of a target gene via expression of a coregulated reporter pBI L 9 gone of p BI Tet gene of interest PminCMV gene
44. ight Vector 6263 1 631059 pTRE Tight EGFP Vector 6264 1 631060 pTRE Tight DsRed2 Vector 6265 1 631061 pLP TRE2 Acceptor Vector 6348 1 631016 Regulator Vectors pTet Off Vector K1620 A 631017 pTet On Vector K1621 A 631018 VP16 Minimal Domain Vector Set K1625 1 631019 pTet tTS Vector 6248 1 631011 Selection Markers pTK Hyg Vector 6153 1 631750 pPUR Vector 6156 1 631601 Linear Hygromycin Marker 6202 1 631625 Linear Puromycin Marker 6203 1 631626 Antibodies and Sequencing Primers VP16 Polyclonal Antibody Affinity Purified 3844 1 631209 pTRE Sequencing PCR Primers 9131 1 631104 pTRE2 Sequencing PCR Primers 9130 1 631103 Protocol PT3001 1 www bdbiosciences com BD Biosciences Clontech Version PR33678 35 Tet Systems User Manual XI Related Products continued Retroviral Expression Systems BD RevTet Off System BD RevTet On System BD Creator Compatible RevTet Off System BD Creator Compatible RevTet On System pRevTet Off Vector pRevTet On Vector pRevTet Off IN Vector pRevTRE Vector pLP RevTRE Packaging Cell Lines Adenoviral Expression Systems BD Adeno X M Tet Off System BD Adeno X M Tet On System BD Adeno X M Tet Off System 2 BD Adeno X Tet On System 2 Cell Culture Tet System Approved FBS US Sourced Tet System Approved FBS USDA Approved BD CalPhos Mammalian Transfection Kit BD CLONfectin Transfection Reagent G418 Hygromycin B Doxycycline Puromycin
45. in Biotechnol 3 506 511 Yin D X amp Schimke T 1995 Bcl 2 expression delays drug induced apoptosis but does not increase clonogenic survival after drug treatment in HeLa cells Cancer Res 55 4922 4928 Yin D X Zhu L amp Schimke R T 1996 Tetracycline controlled gene expression system achieves high level and quantitative control of gene expression Anal Biochem 235 195 201 BD Biosciences Clontech www bdbiosciences com Protocol PT3001 1 34 Version PR33678 Tet Systems User Manual Related Products For a complete listing of all BD Biosciences Clontech products please visit www bdbiosciences com clontech Cat New Cat Tet Off and BD Tet On Cell Lines many many See document PT3001 3 for a complete listing Response Vectors pBI Bidirectional Tet Vector 6152 1 631006 pBI G Bidirectional Tet Vector 6150 1 631004 pBI L Bidirectional Tet Vector 6151 1 631005 pBI EGFP Bidirectional Tet Vector 6154 1 632345 pT RE d2EGFP Vector 6242 1 632346 pTRE2 Vector 6241 1 631008 pTRE Myc Vector 6247 1 631010 pTRE2hyg2 Myc Vector 6257 1 631052 pTRE2pur Myc Vector 6261 1 631055 pTRE HA Vector 6249 1 631012 pTRE2hyg2 HA Vector 6256 1 631051 pTRE2pur HA Vector 6259 1 631054 pTRE 6xHN Vector 6246 1 631009 pTRE2hyg2 6xHN Vector 6258 1 631053 pTRE2pur 6xHN Vector 6262 1 631056 pTRE2hyg Vector 6255 1 631014 pTRE2pur Vector 6254 1 631013 pTRE T
46. ing luciferase from the wild type CMV promoter Well characterized inducer In contrast to the inducer used in other systems such as in the ecdysone system Tc and Dox are inexpensive well characterized and yield highly reproducible results Activation of a promoter rather than repression to control expression To completely shut off transcription repression based systems require very high and difficult to attain levels of repressor to ensure 10096 occupancy of the regulatory sites Even if suitably high levels of repressor can be obtained the presence of high repressor levels makes it difficult to achieve rapid high level induction Yao et al 1998 For a more complete discussion of the advantages of activation versus repression see Gossen et al 1993 In contrast to the heterologous Tet Systems homologous systems based on eukaryotic regulatory elements are subject to one or more of the following problems Inducing stimulus is pleiotropic i e the gene of interest is not the only gene affected by the inducing stimulus 15 removal of 1 ug ml Dox addition of 1 ug ml Dox Luciferase activity RLU x 103 Time hr Figure 3 Luciferase expression is rapidly induced in a BD Tet Off cell line in response to removal of Dox The CHO K1 EGFP Luc Tet Off control cell line expresses the tTA and contains a stably integrated copy of the firefly luciferase gene under control of the TRE Luciferase acti
47. intenance of this site Ausubel F M Brent R Kingdom R E Moore D M Seidman J G Smith J A amp Struhl K eds 1995 Current Protocols in Molecular Biology John Wiley amp Sons NY Baron U Freundlieb S Gossen M amp Bujard H 1995 Co regulation of two gene activities by tetracycline via a bidirectional promoter Nucleic Acids Res 23 3605 3606 Baron U Gossen M amp Bujard H 1997 Tetracycline controlled transcription in eukaryotes novel transactivators with graded transactivation potentials Nucleic Acids Hes 25 2723 2729 Cunningham S M Cunningham M D Zhu L amp Kain S 1997 Determination and correlation of expression levels of luciferase and EGFP using the tetracycline controlled gene expression system and fluorescence imaging Neuroscience Abs 23 647 Freshney R 2000 Culture of Animal Cells Fourth Edition Wiley Liss NY Freundlieb S Schirra M ller C amp Bujard H 1999 A tetracycline controlled activation repression system with increased potential for gene transfer into mammalian cells J Gene Med 1 4 12 Gossen M Bonin A amp Bujard H 1993 Control of gene activity in higher eukaryotic cells by prokaryotic regulatory elements Trends Biochem Sci 18 471 475 Gossen M Bonin A L Freundlieb S amp Bujard H 1994 Inducible gene expression systems for higher eukaryotic cells Curr Opin Biotechnol 5 516 520 Gossen M amp B
48. ion in the Tet On System It encodes a transcriptional silencer tTS that blocks transcription of genes under control of the TRE in the absence of Dox pTet tTS is ideal for regulated expression of toxic genes or other applications that require extremely low level basal expression Alternatively use the pTRE Tight response plasmid 631059 which contains a modified TRE element that can minimize basal expression in certain cell lines See Appendix A for additional information on these vectors H Retroviral Tet Expression The Tet Systems also in a retroviral format The BD RevTet System allows you to stably introduce the elements of the Tet System into virtually any mitotically active cell with high efficiency For more information visit the Tet Systems product page at www bdbiosciences com clontech to download a copy of the BD RevTet User Manual PT3223 1 I Adenoviral Tet Expression The Tet gene expression system is also available in an adenoviral version BD Adeno X Tet Off and BD Adeno X Tet On Expression Systems utilize adenoviral gene transfer to infect dividing and nondividing mamma lian cells for transient regulated expression For further details visit our Tet Systems product page at www bdbiosciences com clontech to obtaina copy of the BD Adeno X Tet Off and Tet On User Manual 496 1 Protocol PT3001 1 www bdbiosciences com BD Biosciences Clontech Version PR33678 Tet Systems User Manual
49. ith 1 2 ug of pTRE2hyg Luc or another reporter vector using the desired transfection method Decrease the amount of DNA if performing liposome mediated trans fection Split into two wells of a six well plate Add Dox 1 2 ug ml to one of the two wells from step 3 Incubate the transfected cells for 48 hr Assay for induction Luciferase Assay calculate fold induction For Tet Off Fold induction Dox RLU Dox RLU For Tet On Fold induction Dox RLU Dox RLU EGFP Assay select clones by flow cytometry Alternatively screen for expression by fluorescence microscopy Select clones with the highest fold induction highest expression with lowest background for propagation and further testing In general only select clones that exhibit gt 20 fold induction Freeze stocks of each clone as soon as possible after expanding the culture Note Some researchers may desire to confirm the presence of the tTA and rtTA regulatory proteins in stable Tet lines by Western analysis with the VP16 Polyclonal Antibody 631209 Use of these antibodies only verifies the presence of tTA or rtTA it does not reveal the functional inducibility of these cell lines Furthermore and rtTA expression in stable cell lines may be below levels detectable by Western blotting High levels of tTA or rtTA are not required for good induction and in fact overexpression of tTA can be toxic to cells Therefore Western analysis sho
50. ll Protocol Overview Figure 4 provides an overview for creating double stable Tet Off or Tet On cell lines which contain integrated copies of the regulatory and response vector the ultimate goal in establishing the Tet System For more detailed flow charts of each of the transfection procedures see Figure 7 Section VIII and Figure 8 Section IX If you have purchased a premade Tet Off or Tet On Cell Line from BD Biosciences Clontech you need only perform the second transfection with your pTRE Gene X construct Important note on simultaneous versus consecutive transfections In general we recommend that you do not attempt to save time by cotransfecting the regulator and response plasmids Cotransfected plas mids tend to cointegrate into the chromosome and enhancer elements from the CMV promoter on the regulator plasmid pTet Off or pTet On can induce basal expression of Gene X Furthermore cotransfection prevents comparison of multiple clones since differences in induction or absolute expression could be due to clone to clone variation in tTA or rtTA expres Sion In contrast consecutive transfections have several advantages Most importantly the response plasmid generally will not cointegrate with the regulator and you can select a double stable cell line that gives very low to no background expression of Gene X Furthermore once you have developed a suitable Tet Off or Tet On cell line it provides a proven genetic background int
51. mit selection of a double stable tet responsive cell line that co expresses two genes pBI G pBI L and pBI EGFP can be used to indirectly monitor expression of a gene of interest for which there is no direct or convenient assay These vectors express p galactosidase luciferase or EGFP enhanced green fluorescent protein as the reporter gene located on one side of the TRE Gene X can be expressed at the same time as the reporter when cloned into the MCS flanking the otherside ofthe TRE When screening double stable cell lines Section IX D you can monitor expression of the reporter from the vector that also simulta neously expresses the gene of interest Expression levels of the gene of interest can be inferred from reporter gene expression in response to Tc or Dox The Vector lacks reporter sequences and instead contains two separate MCSs in opposite orientation driven by two identical inducible promoters pBI allows for co expression of two genes of interest in the same cell For instance the interaction of two proteins or two subunits of a complex protein can be investigated by simultaneous expression in Visit www bdbiosciences com clontech for complete vector information Phi 1 Figure 16 The pBl expression cassette Two genes either two genes of interest a gene of interest and a reporter or two reporters can be expressed simultaneously from the promoter Reporters are firefly luciferase B g
52. n Systems In E coli the Tet repressor protein TetR negatively regulates the genes of the tetracycline resistance operon on the Tn 70 transposon TetR blocks transcription of these genes by binding to the tet operator sequences fetO in the absence of Tc TetR and tetO provide the basis of regulation and induction for use in mammalian experimental systems The first critical component of the Tet Systems is the regulatory protein based on TetR In the Tet Off System this 37 kDa protein is a fusion of amino acids 1 207 of TetR and the C terminal 127 a a of the Herpes simplex virus VP16 activation domain AD Triezenberg et al 1988 Addition of the VP16 domain converts the TetR from a transcriptional repressor to a transcriptional activator and the resulting hybrid protein is known as the tetracycline controlled transactivator tT A tTA is encoded by the pTet Off regulator plasmid which also includes a neomycin resistance gene to permit selection of stably transfected cells The Tet On system is similar to the Tet Off system but the regulatory protein is based on a reverse Tet repressor rTetR which was created by four amino acid changes in TetR Hillen amp Berens 1994 Gossen et al 1995 The resulting protein rtTA reverse tTA is encoded by the pTet On regulator plasmid which also contains a neomycin resistance gene The second critical component is the response plasmid which expresses a gene of interest Gene X under
53. n cell line you will need to perform the entire procedure outlined above If you are starting with one of our premade Tet Off or Tet On Cell Lines only perform the second stable transfection Protocol 4 PT3001 1 www bdbiosciences com BD Biosciences Clontech Version PR33678 13 Tet Systems User Manual lll List of Components Store all mammalian cell lines in liquid nitrogen 196 C Store all plasmids and Fetal Bovine Serum at 20 C Visit our Tet Systems product page www bdbiosciences com clontech for a current list of cell lines and products available for the Tet Systems BD Tet Off Gene Expression System 630921 or K1620 1 20 ul pTet Off Vector 0 5 ug ul 20 so pTRE2hyg Vector 0 5 ug ul 20 pTRE2hyg Luc Vector 0 5 ug ul 0 5 ml CHO AA8 Luc Tet Off Control Cell Line 1 x 10 cells 50 ml Tet System Approved Fetal Bovine Serum pTRE2hyg Vector Information Packet PT3521 5 e Tet Cell Lines Protocol at a Glance PT3001 2 List of Available Tet Cell Lines PT3001 3 BD Tet On Gene Expression System 630922 or K1621 1 20 ul pTet On Vector 0 5 ug l 20 ul pTRE2hyg Vector 0 5 ug l 20 pTRE2hyg Luc Vector 0 5 ug ul 0 5 ml CHO AA8 Luc Tet Off Control Cell Line 1 x 108 cells 50 ml Tet System Approved Fetal Bovine Serum pTRE2hyg Vector Information Packet PT3520 5 e Tet Cell Lines Protocol at a Glance PT3001 2 Listof Available Tet Cell Lines PT30
54. nding of or rTetR to the tetO sequences Genes inserted into one of the sites in the MCS will be responsive to the tTA and rtTA regulatory proteins in the Tet Off and Tet On systems respectively Note that the cloned insert must be in frame with the tag and need not have an ATG or Kozak sequence as these are provided at the start of the tag The tagged fusion protein can be efficiently detected and purified using antibodies and resins optimized against the different markers Complete sequence informa tion is provided in the pTRE Myc HA and 6xHN Vector Information Packets pTRE Myc PT3398 5 pTRE HA PT3462 5 pTRE 6xHN PT3463 5 Protocol 4 PT3001 1 www bdbiosciences com BD Biosciences Clontech Version PR33678 45 Tet Systems User Manual Appendix A Vector Information continued SV40 TRE2 poly Ay Painem Pur Hyg hCMV 1 Em MCS pTRE2Marker Myc amp 6xHN B globin polyA w tag sequence il c Myc HA or 6xHN epitope tag ATG XXX XXX XXX CTT ATG GCC ACT GAC GCG TTG CTA GCG CTG GAA GCT TAT TTG CGG CCG CGT CGA TAT C Mlul Nhe Clal Notl EcoR V Figure 13 pTRE2hyg2 Myc HA amp 6xHN and pTRE2pur Myc HA amp 6xHN composite vector map and multiple clone site MCS These tagged pTRE vectors contain a protein tag sequence followed by an MCS immediately downstream of t
55. necessary to optimize parameters such as cell density the amount and purity of the DNA media conditions and transfection time Once optimized these parameters should be kept constant to obtain reproducible results If cotransfection is required to create a stable cell line with your pTRE vector we recommend cotransfection with Linear Hygromycin Marker 631625 or Linear Puromycin Marker 631626 These markers are short purified linear DNA fragments comprised of the marker gene an SV40 promoter and the SV40 polyadenylation signal Because of their small size these markers are highly effective at generating stable transfectants Alternatively you can use pTK Hyg Vector 631750 or pPUR Vector 631601 Note If you are using a selection vector other than a Linear Selection Marker pTK Hyg or pPUR the promoter should not contain an enhancer element If it does cointegration of the response and selection plasmids may lead to high background expression of Gene X in the uninduced state BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 16 Version PR33678 Tet Systems User Manual IV Additional Materials Required continued For regulation of gene expression Doxycycline 631311 Dilute to 1 2 mg ml in H O Filter sterilize aliquot and store at 20 C in the dark Use within one year Tetracycline hydrochloride Sigma T3383 Dilute to 1 mg ml in 70 ethanol Filter sterilize aliquot and store at
56. nes and can be overcome simply by screening more clones Perform a dose response curve similar to the experiments described in Section VII A The kinetics of induction are dependent on the stability of the mRNA and protein It may take some time before stably expressed proteins accumulate to equilibrium levels Refer to the results seen in Figures 1B 3 and 6 Protocol 4 PT3001 1 www bdbiosciences com BD Biosciences Clontech Version PR33678 31 Tet Systems User Manual IX Development of Double Stable Cell Lines continued Loss of regulation On occasion well characterized double stable cell lines can lose their responsiveness to Tc or Dox This can occur after changing lots of calf or fetal bovine serum and appears to be due to contamination of some lots of serum with Tc If you observe a sudden loss of responsiveness check your serum by performing a dose response curve as described in Section VII A You can also try replating and washing the cells 3 hr later to remove any residual antibiotic that may be interfering with induction control Rennel amp Gerwins 2002 Loss of regulation can also be due to switching off or methylation of the viral promoter It is recommended that you subclone and freeze stocks of your cells at various stages Toxicity of the VP16 activation domain Some researchers have inquired about the possible toxic effects of expressing the VP16 AD in mammalian cells In our experience and that of the Bujard labo
57. o which you can introduce many different response plasmids BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 12 Version PR33678 Tet Systems User Manual ll Protocol Overview continued Perform pilot experiments Section VII 3 weeks Host cell line FIRST STABLE TRANSFECTION Section VIII 2 months Neo pTet Off pTet On Regulatory Transfect with regulator plasmid plasmid pTet Off or pTet On Select G418 resistant clones 20 Screen by transient transfections with pTRE2hyg Luc for clones with low background and high Tc or Dox dependent induction Y BD Tet Off or BD Tet On cell line Premade lines are available from BD Biosciences Clontech SECOND STABLE TRANSFECTION Section IX 2 months Transfect with response plasmid cotransfect with Linear Marker or pTK Hyg or pPUR if necessary pTRE Hyg Pur pTRE2hyg ToM or pTRE Tight Response Select hyg or puro resistant clones plasmid Screen by a gene specific assay for clones with low background and high Tc or Dox dependent induction of Gene X Double stable BD Tet Off or BD Tet On cell line Figure 4 Overview of developing Tet Off and Tet On and double stable Tet Off and Tet On cell lines To use the Tet Gene Expression Systems you will need to make a double stable Tet cell line as outlined above If you are starting with your ow
58. of interest Protocol PT3001 1 Version PR33678 www bdbiosciences com BD Biosciences Clontech 37 Tet Systems User Manual Appendix A Vector Information continued Table BD Tet Off and Tet On Vector alignment continued Applications RevTet Basic Vectors RevTet Off 508 H Wt Pray VP16 Regulator vector for use in RevTet Off P System pRevTet On H YH H E 3 TR Regulator vector for use in RevTet On System pRevTRE 508 H H HygR TRE gene of interest 3 tTR Response vector for use in either RevTet Off or RevTet On Systems RevTet Accessory Vectors pRevTet Off IN s LTR H tetR VP16 IRES Neo 3 LTR Can be used for quickly establishing BD Biosciences Clontech 38 www bdbiosciences com a Tet Off cell line Protocol 4 PT3001 1 Version PR33678 Tet Systems User Manual nued ti Ion con Vector Informati Appendix A eg A H093 91992 II PUH 2g x uiejoJd oAJN oAN Andz3u 19 A 59093 9199 Il PputH VG upfuoJDAux utejoJd oAJN oAW eBAuz3u1d 9ee 9 9A 38 Ld G G Hweg G S euou eseJeJon o AIN onT 9 AW 3u1d 10 998 g0 0 9094 8282 euou X uie1oJd o AN oAAWN 3u1
59. ratory and the many other labs that have successfully used the Tet system this has notbeena problemin tissue culture Like other transcription factors the tTA regulator does not have to be expressed at high levels in order to give very high level expression of the genes it regulates i e genes encoded on the response plasmid For example Gossen and Bujard have characterized HeLa Tet Off cell lines that contain 6 000 10 000 molecules of tTA per cell and give 105 fold induction of the Tet regulated genes pers comm For in vivo applications however it may be preferable to use the VP16 Minimal Domain Vectors which are tolerated at higher intracellular concentrations and allow activation over different ranges See Appendix A for more information BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 32 Version PR33678 Tet Systems User Manual X References You can access an extensive Tet System bibliography from the Tet Systems product page at www bdbiosciences com clontech BD Biosciences Clontech s Tet Systems were developed in cooperation with Dr Bujard and his colleagues at the Center for Molecular Biology in Heidelberg ZMBH Additional background information on Tet regulated gene expression systems is available at the site maintained by Dr Bujard s laboratory http www zmbh uni heidelberg de bujard homepage html Please note that BD Biosciences Clontech is not responsible for the information on or the ma
60. re 5 different lots of FBS exhibit significant variation in their effect on Tet System expression presumably due to the widespread use of tetracy clines in the diet of cattle The gt 10 000 fold induction of luciferase in CHO AA8 Luc Tet Off Control Cells in response to Tc or Dox is highly reproducible If you see a significantly lower level of induction 100 1 000 fold or less this may suggest that your serum contains Tc This test should be repeated with each different lot of serum Alterna tively use Tet System Approved FBS 631101 or 631106 which has been functionally tested and shown to not inhibit the full range of induction possible with the Tet System cell lines 15 x 10 10 x 10 5 Fold induction 5x10 4 Tet System Other commercially Approved FBS available FBS Figure 5 Fold induction of luciferase activity in different lots of FBS The CHO AA8 Luc control cell line was grown in media prepared with different lots of FBS Average uninduced expression level 0 21 RLU 21 S D 0 07 maximum expression levels varied from 123 to 3 176 RLU Protocol 4 PT3001 1 www bdbiosciences com BD Biosciences Clontech Version PR33678 21 Tet Systems User Manual VII Pilot Experiments continued Procedure 1 Plate 6 aliquots of 0 5 x 105 CHO AA8 Luc Tet Off cells each into 5 ml of complete alpha MEM culture medium in 6 well culture dishes 2 To titrate Tc add Tc to final concentrations of 0 1 x
61. shes for viable cells every two days For selecting stable transfectants use a plating density that allows the cells to reach 80 confluency before massive cell death begins at about day 5 This is the cell density at which cells should be plated for selection of stable transfectants For HeLa cells we have found 2 x 109 cells 10 cm dish to be a good plating density Protocol PT3001 1 www bdbiosciences com BD Biosciences Clontech Version PR33678 23 Tet Systems User Manual VII Pilot Experiments continued C optional Test Potential Host Cells by Transient Transfection with pTRE2hyg Luc and pTet Off or pTet On Tet expression systems have been established in numerous cell lines including HeLa CHO MCF7 HEK 293 and HepG2 However the system may not be compatible with every cell type Performing a transient expres sion assay with pTet Off or pTet On and pTRE2hyg Luc may provide a quick indication of whether or not the Tet systems will work in a particular cell line This test is not necessary if you have purchased a premade Tet Off or Tet On Cell Line You shouldtransfect cells using varying ratios of pTet Off Onto pT RE2hyg Luc For example try plet Off On pTRE2hyg Luc 1ug 1 1 10 ug 10 ug 1ug Important Note Fold induction levels are almost always lower in transient assays than in properly screened stable and double stable cell lines For example the Saos 2 Tet Off Cell Line exhibits 40 fol
62. sin EDTA Trypsin Sigma 73924 Dulbecco s phosphate buffered saline DPBS Sigma 08662 Cell Freezing Medium with or without DMSO Sigma 6164 or C 6039 Tissue culture plates and flasks available from BD Discovery Labware www bdbiosciences com discovery labware Cloning cylinders or discs PGC Scientific 62 6150 40 45 or 62 6151 12 16 For transient and stable transfections The transient and stable transfections in this protocol can be performed by various methods Reagents will depend on which transfection method you use Although we generally use electroporation for both transient and stable transfections with the Tet Off and Tet On System other methods work well and may be preferable depending on cell type We offer the BD CalPhos Mammalian Transfection Kit 631312 and BD CLONfectin Transfection Reagent 631301 for high efficiency calcium phosphate or liposome mediated transfections respectively The efficiency of transfection for different cell lines may vary greatly A method that works well for one host cell line may be inferior for another Therefore when working with a cell line for the first time you may want to compare the efficiencies of several transfection protocols You can trans fect the host cell line with a noninducible reporter expression vector such as pCMVB 631719 or pEGFP N1 632318 and assay for reporter gene activity After a method of transfection is chosen it may be
63. site at Position 871 This site can be used to distinguish pTet Off from pTet On pTet Off expresses the tTA tet transactivator regulator protein from the strong immediate early promoter of cytomegalovirus Poyy pTet On expresses the rtTA reverse tTA which contains four amino acid mutations as marked on the map In addition there are several silent mutations in pTet On In all other respects the vectors are identical BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 42 Version PR33678 Tet Systems User Manual Appendix A Vector Information continued Xhol MCS 470 537 P TRE svao Princem uis 470 480 490 500 510 GGATCCTCTAGTCAGCTGACGCGTGCTAGCGCGGCCGCATCGAT Pvull Nhel Notl Clal 514 520 530 pTRE2hyg poly 5 3 kb B albin Xh l AAGCTTGTCGACGATATCTCTAGA Sal EcoRV Xhol EcoRI 50 MCS 470 537 SV40 polyA TRE minCMV P Puro hcMv i EcoRI 79 480 490 500 510 76 GGATCCTCTAGTCAGCTGACGCGTGCTAGCGCGGCCGCATCGAT u 2 Nhel Noti Xho globii 3759 5 1 kb Pgo ni 5 520 530 AAGCTTGTCGACGATATCTCTAGA EcoR V Pul 3142 Figure 10 pTRE2hyg and pTRE2pur vector maps and MCSs Both response vectors contain an MCS immediately downstream of the Tet responsive P cyy promoter P cmv 1 contains the Tet response element TRE whic
64. sponse plasmid or cotransfect pTRE Gene X or pBl Gene X with a Linear Marker Select in presence of hygromycin or puromycin Tc or Dox should be included in the medium when establishing double stable Tet Off cell lines Isolate at least 30 hygromycin puromycin resistant clones e e gt OPTIONAL Confirm presence of integrated pTRE Gene X in clones by PCR Screen by a gene specific assay for clones with Low background of Gene X High induction of Gene X Possible assays Western blot using an antibody to Protein X RT PCR using Gene X primers Northern blot with Gene X probe Functional assay for Protein X Y Reporter activity EGFP D galactosidase or luciferase on pBl vector Double stable Tet Off or Tet On cellline Freeze stocks of double stable cell lines Figure 8 Flow chart for developing double stable Tet cell lines Protocol PT3001 1 www bdbiosciences com BD Biosciences Clontech Version PR33678 29 Tet Systems User Manual IX Development of Double Stable Cell Lines continued 4 Allow cells to divide twice 24 48 hr then add the appropriate 6 T selection agent hygromycin or puromycin to the optimal concentration determined in Section VII For hygromycin the range is generally 200 400 ug ml and for puromycin it is 1 5 ug ml For Tet Off cells only When establishing a double stable Tet Off cell line we r
65. tics of induction and high absolute levels of gene expression Thus for most purposes there is no inherent advantage of using one system over the other With the Tet Off system it is necessary to keep Tc or Dox in the medium to maintain the native off state Because Tc and Dox have relatively short half lives see below you must add Tc or Dox to the medium atleast every 48 hours to suppress expression of Gene X Conversely in the Tet On system the native off state is maintained until induction For this reason Tet On may be more convenient in transgenic applications because you need only add Dox to the animals diet when induction is desired E Tetracycline vs Doxycycline The Tet On System is only responsive to Dox not Tc Gossen amp Bujard 1995 Incontrast Tet Off systems respond equally well to either Tc or Dox We recommend that you use Dox for all Tet System experiments in part because a significantly lower concentration of Dox is required for complete activation or inactivation 0 01 1 ug ml Doxvs 1 2 ug ml Tc In both systems the antibiotics are used at concentrations far below cytotoxic levels for either cell culture or transgenic studies In addition Dox has a longer half life 24 hours than Tc 12 hours Thus for the Tet Off System you may prefer to use Dox for long term maintenance of antibiotic levels and switch to Tc in preparation for induction Other Tc derivatives have been used successfully as th
66. tion near an enhancer may result in high basal expression of Gene X whereas other insertion sites may result in suboptimal induction To find the clone with the highest induction and lowest background we recommend that you grow and analyze as many clones as possible In general test at least 30 clones We have screened as many as 100 clones to obtain one that exhibits suitably high induction and low background IMPORTANT you are not using pTRE2hyg pTRE2pur or another response vector bearing a mammalian selection marker skip the steps below and use the cotransfection protocol in Section IX C 1 Grow cells to 80 confluency in complete medium or to a density appropriate for your transfection method 2 Transfect cells with pTRE2hyg Gene X or pTRE2pur Gene X Note If desired the plasmids can be linearized by digestion with a restriction enzyme check the Vector Information Packets provided with each vector for appropriate restriction sites 3 Plate transfected cells in ten 10 cm culture dishes each containing 10 ml of the appropriate complete medium at the optimal density determined in Section VII BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 28 Version PR33678 Tet Systems User Manual IX Development of Double Stable Tet Cell Lines continued Insert Gene X into pTRE2hyg pur another pTRE variant or a pBl vector pTRE2hyg pur Transfect Tet Off or Tet On cell line with pTRE2hyg pur Gene X re
67. tocol PT3001 1 www bdbiosciences com BD Biosciences Clontech Version PR33678 33 Tet Systems User Manual X References continued pTRE Tight Vectors 2003 Clontechniques XVIII 2 10 1 1 Rennel E amp Gerwins P 2002 How to make tetracycline regulated transgene expression go on and off Anal Biochem 309 79 84 Resnitzky D Gossen M Bujard amp Reed S 1994 Acceleration of the G1 S phase transition by expression of cyclins D1 and E using an inducible system Mol Cell Biol 14 1669 1679 Sambrook J Fritsch E F amp Maniatis T 1989 Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Cold Spring Harbor NY Triezenberg S J Kingsbury R C amp McKnight S L 1988 Functional dissection of VP16 the trans activator of herpes simplex virus immediate early gene expression Genes Devel 2 718 729 Witzgall R O Leary E Leaf A Onaldi D amp Bonventre J V 1994 The Kruppel associated box A KRAB A domain of zinc finger proteins mediates transcriptional repression Proc Natl Acad Sci USA 91 4514 4518 Yao F Svenjo T Winkler T Lu M Eriksson C amp Eriksson E 1998 Tetracycline repressor tetR rather than the tetR mammaiian cell transcription factor fusion derivatives regulates inducible gene expression in mammalian cells Hum Gene Ther 9 1939 1950 Yarronton G T 1992 Inducible vectors for expression in mammalian cells Curr Op
68. ujard H 1992 Tight control of gene expression in mammalian cells by tetracycline responsive promoters Proc Natl Acad Sci USA 89 5547 5551 Gossen M amp Bujard 1993 Anhydrotetracycline a novel effector for tetracycline controlled gene expression systems in higher eukaryotic cells Nucleic Acids Hes 21 4411 4412 Gossen M amp Bujard H 1995 Efficacy of tetracycline controlled gene expression is influenced by cell type BioTechniques 89 213 215 Gossen M Freundlieb S Bender G Muller G Hillen W amp Bujard H 1995 Transcriptional activation by tetracycline in mammalian cells Science 268 1766 1769 Harkin D P Bean J M Miklos D Song Y H Truong V B Englert C Christians F C Ellisen L W Maheswaran S Oliner J D Haber D A 1999 Induction of GADD45 and JNK SAPK dependent apoptosis following inducible expression of BRCA1 Cell 97 575 586 Hillen W amp Berens C 1994 Mechanisms underlying expression of Tn10 encoded tetracycline resistance Annual Rev Microbiol 48 345 369 Kozak M 1987 An analysis of 5 noncoding regions from 699 vertebrate messenger RNAs Nucleic Acids Res 15 8125 8148 Li X Zhao X Fang Y Jiang X Duong T Huang C C amp Kain S R 1998 Generation of destabilized enhanced green fluorescent protein as a transcription reporter J Biol Chem 273 34970 34975 Linear Selection Markers 2003 Clontechniques XVIII 2 11 Pro
69. uld NOT substitute for the functional screen Protocol 4 PT3001 1 www bdbiosciences com BD Biosciences Clontech Version PR33678 27 Tet Systems User Manual IX Development of Double Stable Tet Cell Lines A Test pTRE Gene X by Transient Transfection into a BD Tet Off or Tet On Cell Line Prior to establishing your double stable Tet Off or Tet On cell lines you should test your pTRE Gene X or pBI Tet Vector construct for functional ity Transiently transfect pTRE Gene X into the cell line created in Section VIII or the premade BD Biosciences Clontech Tet Cell Line If you are not using a pBl Vector or one of the tagged vectors pTRE Myc or 6xHN you will need to design a gene specific assay to test for the induction of Gene X Examples of gene specific assays that can be used include Western blot with an antibody to Protein X RT PCR using Gene X primers Be sure you can discriminate PCR products generated from genomic DNA from true RT PCR products Northern blot with Gene X probe Functional assay for Protein X B Stably Transfect and Select Double Stable Cell Lines Figure 8 The next step is to stablytransfect the stable or premade Tet cell line with your pTRE Gene X construct The goal is to generate a cell line that gives low background and high expression of Gene X when tested in Section IX D Both expression levels and induction of Gene X can be profoundly affected by the site of integration Inser
70. vity was continuously monitored with a fluorescent imaging plate reader FLIPR Molecular Devices Corp after addition or removal of 1 ug ml Dox from the culture medium Cunningham et al 1997 BD Biosciences Clontech www bdbiosciences com Protocol PT3001 1 8 Version PR33678 Tet Systems User Manual Introduction continued Itcan be very difficult to distinguish specific from nonspecific events in an expression system based on homologous regulatory elements This is largely due to the modular nature of eukaryotic promoters which interact with a variety of transcription factors that are in turn involved in the regulation of many promoters and or enhancers Most of the commonly used eukaryotic promoters are too leaky to maintain the gene of interest in the fully repressed off state limiting their usefulness for expressing toxic proteins Maximal level of induction is usually not very high Thus of the systems described to date only the Tet Systems exhibit tight on off regulation absence of pleiotropic effects high induction levels high absolute expression and rapid induction times Gossen et al 1993 1994 D BD Tet Off vs BD Tet On Systems Although the Tet Off system has been studied more extensively than the Tet On system the two systems are truly complementary When properly optimized both systems give tight on off control of gene expression regulated dose dependent induction with similar kine
71. xpression use pTRE Tight Vectors No pleiotropic effects When introduced into mammalian cells the prokaryotic regulatory proteins TetR or rTetR the prokaryotic precur sors to tTA and rtTA act very specifically on their target sequences presumably because these regulatory DNA sequences are nonexist ent in eukaryotic genomes Harkin et al 1999 High inducibility and fast response times With the Tet Systems induction can be detected within 30 minutes Figure 3 using nontoxic levels of inducer Induction levels up to 10 000 fold have been ob served results not shown In contrast other systems for mammalian expression exhibit slow induction up to several days incomplete induction compared to repressor free controls low overall induction often no more than 100 fold and high nearly cytotoxic levels of inducer reviewed by Gossen et al 1993 Yarronton 1992 High absolute expression levels Maximal expression levels in the Tet systems can be higher than expression levels obtained from the CMV promoter or other constitutive promoters For example Yin ef al Protocol PT3001 1 www bdbiosciences com BD Biosciences Clontech Version PR33678 7 Tet Systems User Manual Introduction continued 1996 reported that the maximal level of luciferase expression in HeLa Tet Off cells transiently transfected with pTRE Luc is 35 fold higher than that obtained with HeLa cells transiently transfected with a plasmid express
72. y as 100 clones to obtain one that exhibits suitably high induction and low background 1 Grow cells to 80 confluency in complete medium or to a density appropriate for your transfection method 2 Transfect the pTet On or pTet Off Vector by the desired method Note If desired the regulator plasmid can be linearized by digestion with a restriction enzyme Sca for pTet On Off 3 Plate transfected cells in ten 10 cm culture dishes each containing 10 ml of the appropriate complete medium at the optimal density determined in Section VII 4 Allow cells to divide twice 24 48 hr then add G418 to 400 500 ug ml Note The exact concentration of G418 for selection and the optimal plating density may vary from cell type to cell type and with different lots of G418 See Section VII B 5 Replace medium with fresh complete medium plus G418 every four days or more often if necessary After about five days cells that have not taken up the plasmid should start to die Split the cells if they reach confluency before massive cell death begins After 2 4 weeks isolated colonies should begin to appear 6 Isolate large healthy colonies and transfer them to individual plates or wells Suspension cultures must be cloned using the limiting dilution technique When working with adherent cells at BD Biosciences Clontech we generally isolate clones using cloning cylinders or cloning discs Protocol 4 PT3001 1 www bdbiosciences com BD Bios
73. ycin 631626 resistance April 2003 Clontechniques Additionally response vectors are available that express your protein with a tag to aid in detection and protein purification These vectors provide a way to screen colonies directly for protein expression by Western analysis using readily available antibodies These Vectors are available with or without a mammalian selection marker pTRE Myc Vector 631010 pT RE2hyg2 Myc 631052 and pT RE2pur Myc 631055 encode a c Myc tag which is incorporated at the N terminus of the expressed protein The pTRE HA 631012 pTRE2hyg2 HA 631051 and pTRE2pur HA 631054 Vectors encode an HA hemagglutinin epitope tag at the N terminus of the expressed protein allowing detection of the protein with anti HA antibodies The pTRE 6xHN 631009 pTRE2hyg2 6xHN 631053 and pT RE2pur 6xHN 631056 Vectors express proteins that are fused with six His Asn repeats and allow easy purification of your protein using BD TALON Resin or any other immobilized metal affinity column For users of our BD Creator Gene Cloning and Expression System pLP TRE2 Acceptor Vector allows you to quickly transfer your gene of interest into the Tet Systems pLP TRE2 must be cotransfected with a Linear Selection Marker to create stable lines For more information on the BD Creator System please visit our BD Creator product family page at www bdbiosciences com clontech The pTRE2 Sequencing PCR Primers 631103 c
74. ygromycin test a concentration range with a midpoint of 25 g ml Saos 2 Tet Off cells 630911 exhibit resistance to hygromycin test a concentration range with a midpoint of 800 ug ml b Incubate the cells for 10 14 days replacing the selective medium every four days or more often if necessary c Examine the dishes for viable cells every two days For selecting stable transformants use the lowest concentration that begins to give massive cell death in 5 days and kills all the cells within two weeks For HeLa and CHO cells have found 400 ug ml G418 and 200 ug ml hygromycin to be optimal In mammalian cells the optimal level of puromycin is typically around 1 ug ml 2 Determine optimal plating density BD Biosciences Clontech www bdbiosciences com Protocol 4 PT3001 1 22 Version PR33678 Tet Systems User Manual VII Pilot Experiments continued 107 o Sibling Cell Line w o Tc e CHO AA8 Luc e 5 5 g Luciferase activity arbitrary units log transformed amp Luciferase activity arbitrary units log transformed 102 4 102 4 10 T T T T T 1 10 T T T T T T 1 0 0001 001 01 1 10 001 01 1 1 10 100 1000 10000 Tetracycline ug ml Doxycycline ng ml Figure 6 Dose response curves for the CHO AA8 Luc Control Cell Line Panel A Tc dose response curves forthe CHO AA8 Luc Control Cell Line closed circles and a sibling CHO AA8

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