Tet-On® Advanced Inducible Gene Expression Systems User Manual
Contents
1. User Manual Tet On Advanced Inducible Gene Expression Systems User Manual O Clontech United States Canada 800 662 2566 Asia Pacific 1 650 919 7300 Europe 33 0 1 3904 6880 Japan 81 0 77 543 6116 Cat Nos many Clontech Laboratories Inc PT3898 1 102312 ATakara Bio Company p 1290 Terra Bella Ave Published October 2012 Mountain View CA 94043 Technical Support US E mail tech clontech com www clontech com Tet On Advanced Inducible Gene Expression Systems User Manual Table of Contents k MPUCOCUIGEIONN soos cicccsceeusetceetevevesavcsngecctanteasersetuaxiaureccsnxsvinedeseyeiag tecusoudsueereeussniasveetscdeeunsisesseuraniines 3 Py SUNMA i ara arai E EEE EE EE E EE EN EE 3 B Elements of Tet On Advanced Induction c cccecccccsssccessssccsssccecsscecessecccsssececsssecessssecsssececsseseuseseesaeeas 3 C Benefits of the Tet Advanced Expression Systems c cccscsesssesessesescseeseecscseseeeceesesesseesseeeneeesesneaeeeae 4 D Dox yey Cline sescovessesdstnvevsnseestveves soiatesfunsevatiivassenabelvervevareansy snraveotslbmacbelsu EEN E Ea E 4 I List of System Components sirga iabe aerae sieni aireak 5 Ill Additional Materials Required cc ccecceeceeeeeeeeeeeeeceeaeeeeeeeeeee sees eae aeaeeeseeeeeeeeeeeeeseaaes 5 IV Protocol OVORVIGW iiiivsvatsieiissvindiviveceneiebien EE aaa ins sawesudentdetucsayanversvaavavaravaueles 7 A Grenieral Cell Cite AEE E DEEE haste PEE EREE E A AOR E 72. 631067 g pTRE Tight BI AcGFP1 Cat No 631066 8 pTRE Tight Bl DsRed2 Cat No 631064 g pTRE Tight Bl DsRed Express Cat No 631065 Tet Responsive Expression Vectors with ProteoTuner Protein Control Vectors for the inducible coexpression of a gene of interest with protein destabilization control and a fluorescent protein marker g DD pTRE Cycle1 Cat No 631115 DD pTRE Cycle2 Cat No 631116 TE pTRE Cycle3 Cat No 631117 Figure 8 Tet System vectors For a complete list of vectors and their descriptions visit www clontech com Clontech Laboratories Inc www clontech com Protocol No PT3898 1 ATakara Bio Company Version No 102312 17 Tet On Advanced Inducible Gene Expression Systems User Manual Appendix B Titrating Antibiotics for Selecting Stable Cell Lines Protocol 3 5 days A Protocol Titrating Antibiotics for Selecting Stable Cell Lines Prior to using G418 hygromycin or puromycin to select stably transfected cell lines it is necessary to titrate each selection agent to determine the optimal concentration for your target cell line Also the absolute activity of the antibiotic can vary from lot to lot With HeLa cells for example we have found 400 pg ml G418 and 1 0 pg ml puromycin to be optimal e For selecting stable cell lines with G418 or hygromycin use the lowest concentration that results in wide spread cell death in 5 days and kills all the cells within two weeks e Puromycin sel
3. B Elements of Tet On Advanced Induction Based on the original tetracycline Tc regulated transcriptional transactivators described by Gossen amp Bujard 1992 and Gossen et al 1995 Tet On Advanced is a modified transactivator protein that is optimized for expres sion in mammalian cells and which demonstrates higher sensitivity and fidelity than previous versions Urlinger et al 2000 The inducible promoter Py provides for very low basal expression and tightly controlled induction e The Tet On Advanced transactivator The pTet On Advanced vector constitutively expresses the tetracycline controlled transcriptional transactivator Tet On Advanced Urlinger et al 2000 This engineered protein consists of a mutant coli TetR protein rTetR fused to three minimal F type activation domains derived from the herpes simplex virus VP16 protein Baron et al 1997 Triezenberg et al 1988 In the presence of Dox Tet On Advanced binds to the etO sequences in Pre and activates high level transcription from this inducible promoter The Tet On Advanced coding sequence is fully synthetic and utilizes human codon preferences to increase its expression level and stability in mammalian cells The Pingu inducible promoter This is an inducible promoter that controls transcription of your GOI The P rene COMposite promoter was originally developed as the P promoter in the laboratory of Dr H Bujard ight tet 14 and consists of a modi
4. Cat No 631309 Concentration range for selecting stable cell lines 50 800 pg ml Maintenance of stable cell lines 100 pg ml e Puromycin for selecting double stable Tet On Advanced cell lines transfected with the Linear Puromycin Marker Puromycin is available from Clontech Cat Nos 631305 amp 631306 Concentration range for selecting stable cell lines 0 25 10 pg ml Maintenance of stable cell lines 0 25 pg ml C Transfection Reagents e Xfect is a novel highly efficient and versatile transfection reagent that forms biodegradable nanoparticles and produces superior transfection results for a wide variety of mammalian cell types Cat Nos 631317 amp 631318 The CalPhos Mammalian Transfection Kit is a highly efficient calcium phosphate based transfection system Cat No 631312 D Doxycycline Doxycycline Cat No 631311 is needed for inducing expression of your GOI from the transfected pTRE Tight vector Dilute to 1 2 mg ml in H O Filter sterilize aliquot and store at 20 C in the dark Use within one year E Luciferase Assay A method for assaying luciferase expression is required when testing induction in the U2 OS Luc Tet On Control Cell Line or when using the pTRE Tight Luc vector to screen Tet On Advanced clones Use any standard luciferase assay system and luminometer m F Tetracycline Free Fetal Bovine Serum FBS for Target Cell Culture Many lots of bovine sera are
5. This is due to the far higher copies of the TRE containing plasmid present in transiently transfect ed cells compared to the copy numbers in stable cell lines Freeze stocks of each promising clone as soon as possible after expanding the culture Protocol No PT3898 1 Version No 12 102312 www clontech com Clontech Laboratories Inc ATakara Bio Company Tet On Advanced Inducible Gene Expression Systems User Manual IX Developing the Double Stable Tet On Advanced Inducible Cell Line A Functional Testing of pTRE Tight GOI in the Tet On Advanced Cell Line Prior to establishing the double stable Tet On Advanced cell line for your GOI your pTRE Tight GOI construct should be tested for functionality Transiently transfect your pT RE Tight GOI vector into one or more stable cell lines created in Section VIII D and test for GOI induction with Dox You will need an appropriate gene specific assay to test for induction For example Protocol 2 4 weeks o Attention e Western blotting or immunoprecipitation with an antibody to the GOI protein e RT PCR using GOl specific primers Be sure you can discriminate true RT PCR products from products derived from genomic DNA e Northern blotting with a GOl specific probe e Functional assay for the GOI protein B Protocol Creating the Double Stable Tet On Advanced Inducible Cell Line To generate the double stable Tet On Advanced inducible cell line your customiz
6. User Manual VI Culturing Premade Tet Cell Lines continued C Protocol Freezing Tet Cell Line Cultures Once you have started growing a Tet System cell line either a premade one from Clontech or one of your own cell lines prepare frozen aliquots to ensure a renewable source of cells 1 2 3 4 Trypsinize the desired number of flasks or plates Pool cell suspensions together count cells and calculate total viable cell number Centrifuge cells at 100 x g for 5 min Aspirate the supernatant Resuspend the pellet at a density of at least 1 2x10 cells ml in freezing medium Freezing medium can be purchased from Sigma Cat Nos C6164 amp C6039 or freeze cells in 70 90 FBS 0 20 medium with out selective antibiotics and 10 DMSO Dispense 1 ml aliquots into sterile cryovials Freeze slowly 1 C per min For this purpose you can place the vials in Nalgene cryo containers Nalgene Cat No 5100 and freeze at 80 C overnight Alternatively place vials in a thick walled styrofoam con tainer at 20 C for 1 2 hr Transfer to 80 C and freeze overnight Remove vials from the cryo containers or styrofoam containers the following day and place in liquid nitrogen storage or ultralow temperature freezer 150 C for storage Two or more weeks later plate a vial of frozen cells to confirm viability Vil Luciferase Induction in the U2 OS Luc Tet On Cell Line A Testing Luciferase Induction Before you develop
7. Vector 0 5 ug pl 20 pl pTRE Tight Vector 0 5 pg pl 20 pl pTRE Tight Luc Vector 0 5 pg pl 40 pl Linear Hygromycin Marker 50 ng pl 1 ml U2 OS Luc Tet On Control Cell Line 2 0 x 10 cells tube 50 ml Tet System Approved FBS Product Documents and Manuals available at www clontech com manuals Tet On Advanced Inducible Gene Expression System User Manual PT3898 1 pTet On Advanced Vector Information Packet PT3899 5 pTRE Tight Vector Information Packet PT3720 5 Visit www clontech com for a current list of products and cell lines available for the Tet Systems Ill Additional Materials Required A Mammalian Cell Culture Supplies Culture medium supplies and additives specific for your target cells Culture medium for the U2 OS Luc Tet On Control Cell Line 90 Dulbecco s Modified Eagle s Medium DMEM high glucose 4 5 g L 10 Tet System Approved Fetal Bovine Serum FBS 4 mM L glutamine 100 units ml penicillin optional 100 pg ml streptomycin optional 200 pg ml G418 and 100 pg ml hygromycin Tetracycline free fetal bovine serum FBS see important information below We strongly recommend using Tet System Approved FBS Cat Nos 631101 amp 631106 for culturing target cells Cloning cylinders or discs for isolating colonies of adherent cell lines PGC Scientific Cat No 62 6150 40 45 or Cat No 62 6151 12 16 Cell Freezing Medium with or without DMSO Sigma Cat No C6164 or Cat No C6039 for
8. contaminated with tetracycline Tc or Tc derivatives which can affect basal expression or inducibility in Tet Systems Figure 2 It is critical that the FBS used for cell culture not interfere with Tet responsive Attention eV expression e Tc contaminants will diminish the performance of Tet On Advanced based systems by elevating basal expres sion and reducing fold induction e These problems can be eliminated by using a Tet System Approved FBS Cat Nos 631101 amp 631106 from Clontech These sera have been functionally tested in our Tet Systems and found to be free of contaminating Tc activity 15 x 10 10x 10 Fold induction 5x 10 Tet System Other commercially Approved FBS available FBS Figure 2 Tetracycline activity in bovine sera The CHO AA8 LucTet Off Control Cell Line was grown in media prepared with different lots of FBS Average uninduced expression level 0 21 RLU n 21 S D 0 07 maximum expression levels varied from 123 to 3 176 RLU Protocol No PT3898 1 www clontech com Clontech Laboratories Inc Version No 102312 ATakara Bio Company 6 Tet On Advanced Inducible Gene Expression Systems User Manual IV Protocol Overview PLEASE READ THESE PROTOCOLS IN THEIR ENTIRETY BEFORE STARTING Successful results depend on understanding and performing the following steps correctly A General Cell Culture This user manual provides only general guidelines for mammalian cell culture techniques P
9. freezing Tet On Advanced cell lines and double stable Tet On Advanced cell lines Optional B Antibiotics for Selecting Stable Cell Lines Prior to using antibiotics to select stable cell lines from your transfected cells determine the optimal selection con centration for each antibiotic as described in Appendix A For example the G418 concentration range for selecting stable HeLa cell lines is 400 500 pg ml However each new lot of any selection antibiotic should be titrated e G418 for selecting single stable Tet On Advanced cell lines G418 is available from Clontech Cat No 631307 Note that the effective weight is approximately 0 7 g per gram of powder Make a 10 mg ml stock solution by dissolving 1 g of powder in approximately 70 ml of culture medium without supplements Filter sterilize and store at 4 C Concentration range for selecting stable cell lines 50 800 ug ml Maintenance of stable cell lines 100 pg ml Selection concentration e g HEK 293 HeLa cells 400 500 pg ml Clontech Laboratories Inc ATakara Bio Company www clontech com Protocol No PT3898 1 Version No 102312 5 Tet On Advanced Inducible Gene Expression Systems User Manual lll Additional Materials Required continued B Antibiotics for Selecting Stable Cell Lines cont d e Hygromycin for selecting double stable Tet On Advanced cell lines transfected with the Linear Hygromycin Marker Hygromycin B is available from Clontech
10. tissue culture wells Isolate as many clones as fea sible so that at least 30 clones are available for testing Suspension cultures must be cloned using a limiting dilution technique Culture the clones in a maintenance concentration of G418 100 200 ug ml When they have grown suf ficiently proceed with testing the clones for induction as described in Section VIII D D Protocol Testing Your Tet On Advanced Clones for Induction 1 For each clone to be tested seed 1 3 of the total 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 Divide the remaining 2 3 of the cells between duplicate wells of a second 6 well plate Allow the cells to ad here overnight and transfect each well with pTRE Tight Luc using the amount of DNA appropriate for your preferred transfection method When transfection is complete replace the transfection medium with fresh culture medium and add Dox 0 01 1 0 ug ml to one of the duplicate wells while leaving the second well Dox free Incubate the cells with Dox for 48 hr Assay for luciferase activity and calculate fold induction e g Dox RLU Dox RLU Select clones with the highest fold induction for propagation and further testing i e clones that exhibit gt 20 fold induction NOTE When testing clones via transient transfection you can expect lower fold induction levels than in double stable clones
11. your own Tet On Advanced System we strongly recommend that you perform a Dox dose response curve using the U2 OS Luc Tet On Cell Line This premade double stable Tet On pTRE Luc cell line exhibits gt 500 fold induction of luciferase when cultured in the presence of Dox In addition to providing a hands on experience with a Tet On system this experiment 1 calibrates the effective concencentration of your Dox i e full activation should be achieved with 100 1000 ng ml Dox and 2 verifies that your tissue culture medium and serum are free of tetracycline contamination Luciferase induction in the U2 OS Luc Tet On cell line is highly reproducible If induction is significantly lower than 500 fold or if a high level of basal expression is observed it is possible that your serum is contaminated with Tc B Protocol Inducing Luciferase Expression in U2 OS Luc Tet On Cells 1 After thawing and establishing the cell line plate 5 x 104 U2 OS Luc Tet On cells in a volume of 2 3 ml of Protocol complete culture medium into 8 12 wells of two 6 well culture dishes 2 3 2 Add Dox to a series of wells at final concentrations of 0 1 x 10 1 x 107 0 1 1 0 10 100 and 1000 ng ml days respectively 3 Allow the cells to grow for 48 hr 4 Assay each sample for luciferase activity using any standard luciferase assay Plot your results and compare to Figure 4 page 8 Protocol No PT3898 1 Version No 102312 10 www clont
12. B Establishing the Tet On Advanced System in Target Cells seessseeseeeesessesesresrsresrsrrsrssrersrtnreresreresrerese 7 V Plasmid Propagation and Vector Construction cccccsssssssssseseseseseceeeeeeeeeeeeeeeeseseeeseeaneas 8 A General Molecular Biology Techniques s csccccyetncieevecssedeotscobstsapbescpaerdussednedecyidanseensenrdecevietvensdsenoranss 8 B Plasmid Propagation amp Construction of Your pTRE Tight Vector ccccsccsseseseseeesseseesenseeeseneneeeeees 8 VI Culturing Premade Tet Cell Limes c cccccceceeeeeeeeeeeeeeeesaeaeaeesasaseeessseceeeeeeeeseeeeeeseeseeeseeaeoas 9 A Characteristics of Tet Cell Limes ccccccccscsccssssccssssccssscesssssssssssscvsssssscsssessscsscssssssssssscesssscosssssessessssees 9 B Protocol Starting Tet Cell Cultures from Frozen StockSiiiisrisnieosninai ennei visiini 9 C Protocol Freezing Tet Cell Line Cultures isseire in EE E EE 10 VII Luciferase Induction in the U2 OS Luc Tet On Cell Line cceceeeeeeeeeeeeeeeeeeeeeeeees 10 Ay Testing Luciferase Tnductiois nein nense aee an E erinda coosstinee ENTES SE EES E n 10 B Protocol Inducing Luciferase Expression in U2 OS Luc Tet On Cells s ssssssssssesessererereeereerererrenee 10 VIII Developing a Tet On Advanced Cell Line c cccccccccecsscssssesssseseeseeeeeesseessseeaneeseeeeeeeeeees 11 As SUNAY ea TAE EE EOE EEE EEPE A EEA EE SEE EESE EEE EE 11 B Protocol Pilot Testing Tet Based Inductio
13. Technical Support Telephone 800 662 2566 toll free Telephone 800 662 2566 toll free Fax 800 424 1350 toll free Fax 800 424 1350 toll free Web www clontech com Web www clontech com E mail orders clontech com E mail tech clontech com Notice to Purchaser Clontech products are to be used for research purposes only They may not be used for any other purpose including but not limited to use in drugs in vitro diag nostic purposes therapeutics or in humans Clontech products may not be transferred to third parties resold modified for resale or used to manufacture commercial products or to provide a service to third parties without prior written approval of Clontech Laboratories Inc Your use of this product is also subject to compliance with any applicable licensing requirements described on the product s web page at http www clontech com It is your responsibility to review understand and adhere to any restrictions imposed by such statements Clontech the Clontech logo Cal Phos In Fusion ProteoTuner Stellar Tet Off Tet On and Xfect are trademarks of Clontech Laboratories Inc All other marks are the property of their respective owners Certain trademarks may not be registered in all jurisdictions Clontech is a Takara Bio Company 2012 Clontech Laboratories Inc This document has been reviewed and approved by the Clontech Quality Assurance Department Clontech Laboratories Inc ATakara Bio Company w
14. al_ Hindili Sall EcoRV Xbal Xho Notl Accl Figure 6 Map and MCS of pTRE Tight For a complete vector description refer to the enclosed Vector Information Packet PT3720 5 Luciferase pTRE Tight Luc 4 2 kb sv40 poly A Figure 7 Map of pTRE Tight Luc Protocol No PT3898 1 www clontech com Clontech Laboratories Inc Version No 102312 ATakara Bio Company 16 Tet On Advanced Inducible Gene Expression Systems User Manual Appendix A Tet Vector Information A Tet Advanced Transactivator Plasmids Vectors that express the Tet On Advanced regulator for inducible gene expression in the presence of doxycycline Pomvie Tet On Adv Neo pTet On Advanced Cat Nos 630930 amp 631069 Powe A TeeOmAdv pTet DualON Cat No 631112 Vectors that express the Tet Off Advanced regulator for inducible gene expression in the absence of doxycycline Iai Tet Off Adv Neo pTet Off Advanced Cat Nos 630934 amp 631070 Powie A eCOM AJY pTet DualOFF Cat No 631113 E Tet Responsive Expression Vectors Vectors for the inducible expression of one or two genes of interest Frio pTRE Tight Cat No 631059 Foo ires2 IK pTRE Dual1 Cat No 631114 i aan a pTRE Tight BI Cat No 631068 Vectors for the inducible coexpression of a gene of interest and a fluorescent protein marker Fas ress If pTRE Dual2 Cat No 631112 amp 631113 j K pTRE Tight Bl ZsGreen1 Cat No
15. an cells Curr Opin Biotechnol 3 506 511 Yin D X amp Schimke R 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 Clontech Laboratories Inc ATakara Bio Company www clontech com Protocol No PT3898 1 Version No 102312 15 Tet On Advanced Inducible Gene Expression Systems User Manual Appendix A Tet Vector Information Clontech offers a wide variety of inducible expression vectors designed for use with Tet Expresssion Systems Figure 8 Visit www clontech com for a complete list of currently available vectors The vectors below are supplied with the Tet On Advanced Gene Expression System Cat No 630930 EcoRI Aol 766 Tet On Advanced rtTA28 M2 Ne pTet On Advanced 7 1 kb poya LI Hindill 1988 BamHI 4212 Xhol 4200 Figure 5 Map of pTet On Advanced For a complete vector description refer to the enclosed pTet On Advanced Vector Information Packet PT3899 5 MCS 323 411 Pvul 1985 pTRE Tight 2 6 kb 323 GAATTCGAGCTCGGTACCCGGGGATCCTCTAGTCAGCTGACGCGT EcoRI Kpnl BamHI Pvull Mlul 368 Smal GCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTCTAGA Nhel Eagi Cl
16. appropriate for your transfection method After 12 24 hr transfect them with the pTet On Advanced Vector by your preferred method NOTE Using an alternative Tet On Advanced expression vector such as pTet DualON Cat No 631112 requires that a linear selection marker hygromycin or puromycin be cotransfected along with the vector plasmid Use a vector to marker molar ratio of 20 1 i e 20 fold less marker than plasmid A different marker must be used when transfecting the TRE based expression vector in Section IX When transfection is complete reseed the transfected cells in 10 cm plates in complete culture medium Use the plating density for your cell line that is optimal for G418 selection Appendix B Allow cells to divide twice 24 48 hr then add G418 at the selection concentration that is optimal for your cell line For most cell lines this is usually 400 500 pg ml Replace medium with fresh complete medium plus G418 every four days or more often if necessary Cells that have not taken up the plasmid should begin to die after 5 days Avoid passaging the cells a second time since replating cells under selection may result in plates containing too many colonies for effective colony isolation After 2 weeks G418 resistant colonies should begin to appear When the colonies are large enough to transfer use cloning cylinders or disks to harvest i e pick large healthy colonies and transfer them to individual plates or
17. ard primer 5 X AGGCGTATCACGAGGCCCTTTCGT 3 located at 2577 2600 e Reverse primer 5 TATTACCGCCTT TGAGTGAGCTGA 3 located at 683 660 NOTE Do not use the pTRE or pTRE2 Sequencing Primers These primer sets are incompat ible with pTRE Tight U2 0S Luc Tet On Control Cells 3 000 2 500 2 000 1 500 1 000 Luciferase activity RLU 500 0 0 1 1 0 10 100 1000 Doxycycline ng ml Figure 4 Doxycycline dose response curve for the U2 OS Luc Tet On Control Cell Line Protocol No PT3898 1 www clontech com Clontech Laboratories Inc Version No 102312 ATakara Bio Company 8 Tet On Advanced Inducible Gene Expression Systems User Manual VI Culturing Premade Tet Cell Lines A Characteristics of Tet Cell Lines Clontech premade Tet System Cell Lines such as the U2 OS Luc Tet On Control Cell Line have been developed and functionally tested for use with Tet expression systems and a wide variety of Tet and TRE based vectors See the Certificate of Analysis of each individually purchased cell line for specific information regarding its culture mainte nance and drug resistance Detailed instructions for the use of these cell lines are available in the Tet Systems User Manual PT3001 1 and the Tet Cell Lines Protocol at a Glance PT3001 2 A complete listing of all available cell lines can be found at www clontech com B Protocol Starting Tet Cell Cultures from Frozen Stocks Important Frozen cells should be cu
18. ce it in a 37 C humidified incubator 55 10 CO2 as appropriate for 24 hrs Vz Note For HEK 293 based cell lines we recommend using collagen coated plates or flasks for efficient cultur ing of frozen stocks Vessels coated with compounds other than collagen may also provide suitable growth substrates e g poly L lysine but only collagen has been tested at Clontech Once recovered the cells may be cultured directly on tissue culture plastic However if adherence is poor we recommend using only collagen coated vessels Note For Jurkat and other suspension cultures suspend cells at a density of no less than 2x10 cells ml 5 The next day examine the cells under a microscope If the cells are well attached and confluent they can be passaged for use If the majority of cells are not well attached continue culturing for another 24 hrs Note For HEK 293 based cell lines complete attachment of newly thawed cultures may require up to 48 hrs 6 Expand the culture as needed Note The appropriate selective antibiotic s should be added to the medium after 48 72 hr in culture Maintain stable and double stable Tet Cell Lines in complete culture medium containing a maintenance concentration G418 and or hygromycin as appropriate Typically this is 100 ug ml for each drug Clontech Laboratories Inc www clontech com Protocol No PT3898 1 ATakara Bio Company Version No 102312 9 Tet On Advanced Inducible Gene Expression Systems
19. d in cloning grow a sufficient culture volume of transformed bacteria and purify the plasmid DNA using an appropriate NucleoBond Xtra or NucleoSpin kit see www clontech com or an equivalent purification method 3 Using standard cloning techniques and appropriate directional restriction sites clone your GOI cDNA frag ment in the multiple cloning site MCS of pTRE Tight or your TRE based vector of choice see Appendix A You may also use Clontech s In Fusion technology such as the In Fusion HD PCR Cloning System Cat No 639648 In Fusion allows PCR products to be easily cloned without restriction enzyme digestion or ligation into any linearized vector To use In Fusion you must synthesize PCR primers that are specifically designed for this purpose For more information see the In Fusion HD PCR Cloning System User Manual 4 Perform a midi or maxi scale plasmid DNA preparation for each plasmid that will be transfected into target cells For guaranteed transfection grade plasmid DNA we recommend using NucleoBond Xtra Midi Plus or Maxi Plus Kits Cat Nos 740412 10 and 740416 10 For rapid production of endotoxin free transfec tion grade plasmid DNA use NucleoBond Xtra Midi EF Plus or Maxi EF Plus Kits Cat Nos 740422 10 and 740426 10 Sequencing the GOI Insert in pTRE Tight Following cloning of pTRE Tight GOI plasmid the insertion junctions should be confirmed by sequencing Specific primers for pTRE Tight are e Forw
20. dd Dox 0 01 1 0 pg ml to one of the duplicate wells for each vector ratio being tested Use the second well in each duplicate as an untreated control If multiple wells are available for each ratio test a range of Dox concentrations 3 After 12 24 hr of treatment with Dox harvest the cells and assay for luciferase activity Compare Dox cells to Dox cells to determine fold induction NOTE Due to the very high plasmid copy numbers contained in transiently transfected cells fold induction levels are almost always lower in transient assays than in properly screened stable and double stable clonal cell lines For example the Saos 2 Tet Off Cell Line exhibits 40 fold induction in transient expression assays but stable clones can be isolated that exhibit 6 000 fold induction and have basal expression levels that are indistinguishable from control background expression Therefore an apparent low level of induction is not necessarily a true indication of the inducibility that can be ultimately attained in a particular cell line Clontech Laboratories Inc www clontech com Protocol No PT3898 1 ATakara Bio Company Version No 102312 11 Tet On Advanced Inducible Gene Expression Systems User Manual VIII Developing a Tet On Advanced Cell Line continued Protocol 2 4 weeks Protocol 2 3 days C Protocol Creating a Stable Tet On Advanced Cell Line from Your Target Cell Line 1 Plate target cells at a density
21. ducible transgenic lines Gossen amp Bujard 2002 More than 280 mouse lines have been described that ex press Tet transactivator genes under the control of a variety of tissue specific promoters or that express target genes under control of Tet inducible promoters A list of these mouse lines can be found on the TET Systems website http www tetsystems com support transgenic mouse lines With its greatly increased sensi tivity to Dox the Tet On Advanced System brings additional advantages to the development of inducible transgenic mice This may be particularly helpful when control of gene expression in the brain is required as the presence of the blood brain barrier limits the concentration of Dox that can be attained in the brain D Doxycycline The doxycycline concentrations required for induction with Tet On Advanced Systems are far below cytotoxic levels for either cell culture or transgenic studies Of note Tet On Systems respond only to Dox and not to Tc Gossen amp Bujard 1995 Protocol No PT3898 1 Version No 4 102312 www clontech com Clontech Laboratories Inc ATakara Bio Company Tet On Advanced Inducible Gene Expression Systems User Manual ll List of System Components Store frozen mammalian cell lines in liquid nitrogen 196 C Store all plasmids and Fetal Bovine Serum at 20 C Tet On Advanced Inducible Gene Expression System Cat No 630930 Package Contents 20 pl pTet On Advanced
22. ech com Clontech Laboratories Inc ATakara Bio Company Tet On Advanced Inducible Gene Expression Systems User Manual VIII Developing a Tet On Advanced Cell Line A Summary The first step in establishing the Tet On Advanced Inducible Expression System is creating a Tet On Advanced stable cell line that 1 expresses the Tet On Advanced transactivator 2 demonstrates high levels of Ppp induc tion and 3 exhibits low basal expression from P This Tet On Advanced cell line will be subsequently trans fected with your customized pTRE Tight GOI vector which will ultimately enable your target cells to express your GOI when the cells are treated with Dox For best results we suggest that you use a high efficiency transfection method such as Clontech s Xfect Cat No 631317 or CalPhos Mammalian Transfection Kit Cat No 631312 and optimize the transfection conditions for your target cell type Parameters to be optimized include initial plating density transfection time plating den sity for drug selection G418 concentration for selection etc Once G418 selection has been completed we recommend that you isolate as many clones colonies as possible in Section VIII C Step 7 In general isolate and expand enough colonies to be able to zest at least 30 clones Note that not all picked colonies will survive isolation and expansion While it is possible to identify an optimal clone by screening fewer than 30 clones our experience ha
23. ection occurs more rapidly use a concentration that will kill all cells within 3 4 days e If possible test several plating densities versus each antibiotic concentration If cells become heavily confluent before they begin to die viable clones may be lost if they detach from the plate Also passaging cells while they are under selection is not recommended e IMPORTANT Lot to lot variations in potency exist for all selection drugs so each new lot of antibiotic should be titrated 1 For each antibiotic to be tested plate 2 x 10 cells in each well of a 6 well plate containing 3 ml of the appro priate complete medium plus increasing concentrations of G418 0 50 100 200 400 and 800 pg ml For puromycin add the drug at 0 1 0 2 5 5 0 7 5 and 10 0 ug ml 2 For G418 incubate the cells for 5 10 days or until all cells are dead Examine the dishes for viable cells every two days Replace the selective medium every four days or more often if necessary until the optimal concentration is determined 3 For puromycin incubate the cells 4 7 days Replace medium after 2 days to remove dead cells Appendix C Using pTRE Cycle Vectors to Rapidly Control Protein Levels A Summary Clontech s pTRE Cycle vectors allow precise rapid control of inducible expression by using a multi tiered approach that combines the powerful transcriptional control of the Tet Advanced System with the controlled protein destabi lization of the ProteoT
24. ed pTRE Tight vector or any other TRE based vector is cotransfected along with the selection marker into your Tet On Advanced cell line Stable transfectants are selected using hygromycin or puromycin 1 2 Plate your Tet On Advanced cell line at a density appropriate for your preferred transfection method Combine your customized pTRE Tight vector and either the Linear Hygromycin or Puromycin Marker at a ratio of 20 1 i e 20 fold less linear marker and transfect the Tet On Advanced cells using your preferred method NOTE If the Linear Hygromycin Marker was used to create the Tet On Advanced cell line you must cotransfect the Linear Puromycin Marker with the TRE based vector to create the double stable cell line When transfection is complete seed the transfected cells in 10 cm plates Use complete medium containing an appropriate maintenance concentration of G418 100 200 pg ml Use the plating density for your cell line that is optimal for hygromycin or puromycin selection Appendix B Allow cells to divide twice 24 48 hr time will vary with cell line before adding hygromycin 200 400 pg ml or puromycin 1 10 pg ml to the culture medium Use the drug concentration optimal for your cell line Appendix B Continue drug selection until colonies are visible and all untransfected cells have died Avoid passaging the cells a second time since replating cells under selection may result in plates containing too many coloni
25. erform all steps involv ing cell culture using sterile technique in a tissue culture hood For users requiring more information on mammalian cell culture transfection and creating stable cell lines we recommend the following general reference Culture of Animal Cells 5th Edition by R I Freshney 2005 Wiley Liss NY B Establishing the Tet On Advanced System in Target Cells The general strategy for establishing the Tet On Advanced System is shown in Figure 3 in which target cells are first transfected with pTet On Advanced to create a stable cell line Once a suitable Tet On Advanced cell line clone is identified the cell line is then stably transfected with your customized TRE based vector containing your GOI Transfect target cells with pTet On Advanced Select for stably transfected cells N Pick 230 colonies clones expand and screen for inducibility Carry forward best clone Tet On Advanced cell line a Gol 3 T aL E pas Hygromycin J cell line wi based vector an a linear marker Select for stably or puromycin transfected cells selection Hyg Pur 4 Pick clones expand and screen or sort cells for GOI expression induced by Dox Target cell line containing a Tet On Advanced Inducible Expression System GOI OFF Figure 3 Establishing the Tet On Advanced System in target cells Target cells are transfected with the pTet On Advanced plasmid and selected with G418 to generate a stab
26. es for effective colony isolation Colonies should be visible in 2 4 weeks When colonies are large enough to transfer use cloning cylinders or disks to isolate large healthy colonies and transfer them to individual plates or tissue culture wells Harvest as many clones as feasible so that at least 30 clones are available for testing Suspension cultures must be cloned using a limiting dilution tech nique Culture the clones in medium containing maintenance concentrations of G418 and hygromycin or puromy cin When they have grown sufficiently test the clones for induction as described in Section C Note Working with mixed polyclonal populations of transfected cells rather than selecting for single clones can affect the consistency of induction due to the possible outgrowth of poorly inducing clones as the cells are passaged Clontech Laboratories Inc ATakara Bio Company www clontech com Protocol No PT3898 1 Version No 102312 13 Tet On Advanced Inducible Gene Expression Systems User Manual IX Developing the Double Stable Tet On Advanced Inducible Cell Line C Protocol Screening Your Panel of Double Stable Tet On Advanced Inducible Cell Lines Test individual double stable clones for expression of your GOI in the presence and absence of several concentra tions of Dox 10 1000 ng ml Choose clones that generate the highest overall induction and lowest basal expres Protocol 2 3 sion i e highest fold inducti
27. fied Tet Responsive Element TRE containing 7 direct repeats of the ter operator sequence tetO which is joined to a minimal CMV promoter P cyiya gt Prig Lacks binding sites for endog enous mammalian transcription factors so it is virtually silent in the absence of induction In the presence of Dox Tet On Advanced binds tightly and specifically to Pig GOI Figure 1 and activates transcription of the downstream Dox 3X min VP16 Tet On Advanced Transcription ne Gene of interest Tet On Advanced System Figure 1 Induction in the Tet On Advanced System The Tet controlled transactivator Tet On Advanced is a fusion protein derived from a mutant version of the E coli Tet repressor protein rfetR which is joined to three minimal transcription activation domains from the HSV VP16 protein In the presence of doxycycline Dox Tet On Advanced binds to the tetracycline response element TRE moa in Pig and produces high level transcription of the downstream gene of interest Clontech Laboratories Inc ATakara Bio Company www clontech com Protocol No PT3898 1 Version No 102312 3 Tet On Advanced Inducible Gene Expression Systems User Manual l Introduction continued C Benefits of the Tet Advanced Expression Systems The Tet On Advanced System produces very high maximal expression coupled with extremely low basal promoter activity to yield very high induction levels that are both high
28. ible for the information contained on this website 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 Res 25 2723 2729 Freshney R I 2005 Culture of Animal Cells 5th Edition Wiley Liss New York NY Freundlieb S Schirra Miiller C amp Bujard H 1999 A tetracycline controlled activation repression system with increased potential for gene transfer into mam malian 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 amp Bujard 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 H 1995 Efficacy of tetracycline controlled gene expression is influenced by cell type Bio Techniques 89 213 215 Gossen M amp Bujard H 2002 Studying gene function in eukaryotes by conditiona
29. l gene inactivation Annu Rev Genet 36 153 73 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 amp Haber D A 1999 Induc tion of GADD45 and JNK SAPK dependent apoptosis following inducible expression of BRCA1 Cell 97 575 586 Sambrook J Fritsch E F amp Maniatis T eds 2001 Molecular Cloning A Laboratory Manual Cold Spring Harbor Laboratory Press 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 expres sion Genes Devel 2 718 729 Urlinger S Baron U Thellmann M Hasan M T Bujard H amp Hillen W 2000 Exploring the sequence space for tetracycline dependent transcriptional activa tors Novel mutations yield expanded range and sensitivity Proc Natl Acad Sci USA 97 14 7963 7968 Yao F Svenjo T Winkler T Lu M Eriksson C amp Eriksson E 1998 Tetracycline repressor tetR rather than the tetR mammalian cell transcription factor fu sion derivatives regulates inducible gene expression in mammalian cells Hum Gene Ther 9 1939 1950 Yarronton G T 1992 Inducible vectors for expression in mammali
30. le Tet On Advanced cell line This cell line serves as the host for a TRE based expression vector which is transfected into the Tet On Advanced cell line along with a linear selection marker Hyg or Pur After a second round of drug selection a stable cell line is produced which expresses high levels of the GOI in response to doxycycline Dox Clontech Laboratories Inc ATakara Bio Company www clontech com Protocol No PT3898 1 Version No 102312 7 Tet On Advanced Inducible Gene Expression Systems User Manual V Plasmid Propagation and Vector Construction A General Molecular Biology Techniques Only general information for propagating cloning and purifying plasmid vectors is provided below For users requir ing detailed information on plasmid propagation and cloning we recommend the following laboratory references Current Protocols in Molecular Biology ed by F M Ausubel et al 1995 John Wiley amp Sons NY Molecular Cloning A Laboratory Manual ed by J Sambrook et al 2001 Cold Spring Harbor Laboratory Press NY B Plasmid Propagation amp Construction of Your pTRE Tight Vector 1 To ensure that you have a renewable source of plasmid DNA transform each of the plasmids provided into a suitable E coli host strain e g Stellar Electrocompetent Cells Cat No 636765 See the specific Vector Information Packet supplied with each vector for further DNA propagation details 2 For plasmids to be use
31. ltured immediately upon receipt or as soon as possible thereafter If culturing after shipping is significantly delayed decreased cell viability may result To prevent osmotic shock and maximize Protocol i 2 3 cell survival follow the steps below to begin a new culture from frozen cells days 1 Thaw the vial of cells rapidly in a 37 C water bath with gentle agitation Immediately upon thawing wipe the outside of the vial with 70 ethanol All of the operations from this point on should be carried out in a laminar flow tissue culture hood under strict aseptic conditions Unscrew the top of the vial slowly and using a pipet transfer the contents of the vial to a 15 ml conical centrifuge tube containing 1 ml of pre warmed medium without selective antibiotics e g G418 Mix gently 2 Slowly add an additional 4 ml of fresh pre warmed medium to the tube and mix gently 3 Add an additional 5 ml of pre warmed medium to the tube mix gently Centrifuge at 100 x g for 5 min carefully aspirate the supernatant and GENTLY resuspend the cells in complete medium without selective antibiotics This method removes the cryopreservative and can be beneficial when resuspending in small volumes However be sure to treat the cells gently to prevent damaging fragile cell membranes 4 Mix the cell suspension thoroughly and add to a suitable culture vessel Gently rock or swirl the dish flask to distribute the cells evenly over the growth surface and pla
32. ly sensitive and concentration dependent Advantages over other inducible mammalian gene expression systems are listed below Extremely tight regulation In the absence of induction the Tet On Advanced transactivator shows virtually no residual binding to the TRE in P ie Thus basal expression is extremely low and often undetectable Highly specific The rTetR portion of the Tet On Advanced transactivator bind very specifically to the tetO target sequences of P and does not activate off target cellular genes This high degree of specificity may be due in part to the prokaryotic nature of these components acting within the context of a large eukaryotic genome lacking similar elements Harkin et al 1999 No pleiotropic effects Tc and Dox are prokaryotic antibiotics that have no known effects on eukaryotic cells when used at the concentrations required by the Tet Advanced Systems High inducibility and fast response times In properly screened clones maximal induction of the Tet On Advanced System is often several thousand fold and can be detected within 30 minutes after addition of Dox to the culture medium In contrast other mammalian systems often exhibit slow induction up to several days incomplete induction compared to repressor free controls and low overall induction often no more than 100 fold Other systems may also require high nearly cytotoxic levels of inducer reviewed by Gossen et al 1993 Yarronton 1992 Highest absol
33. n Advanced System in target Cells ccecsescseeeeesesseeenseereseeeetseeeeeetenaes 7 Figure 4 Doxycycline dose response curve for the U2 OS Luc Tet On Control Cell Line wn 8 Figure 5 Map of p let On Advanced sisicessscsisesicescalonesevedciys ia ietenasivessdnsdovedsess eongsipdesiaweacetadiuenesuededyeetss 16 Figure 6 Map and MGS of pURE Tighttss escviciecsseicscssssassentasibabacoevtaveteanteastsauspepneasubas savessbeye IAEE Ein 16 Figure 7 Map of the p TRE Tight Luc Vectors sisisi nara ariaa eii naraenia 16 Figure 8 Tet Systemi Vectors ccssss sacsssnesssnctsssevesssbeusetbesssouns snes snsselnessvaebacnevbsesbaateenessvabeesdesensbecnessshavreneyiens 17 Protocol No PT3898 1 www clontech com Clontech Laboratories Inc Version No 102312 ATakara Bio Company 2 Tet On Advanced Inducible Gene Expression Systems User Manual l Introduction A Summary The Tet On Advanced Inducible Gene Expression System is a tightly regulated and highly responsive system that produces on demand robust expression of your gene of interest GOI in target cells The system is established in target cells by sequentially transfecting them with the provided vectors and selecting stable cell lines Target cells that express the Tet On Advanced transactivator and that also contain an integrated TRE based expression vector e g pTRE Tight will express high levels of your GOI when cultured in the presence of the systems inducer doxycycline Dox Figure 1
34. n in Target Cells siirsiiseissrisresncinseesssncreetsrinesriseriseriiie ises 11 C Protocol Creating a Stable Tet On Advanced Cell Line from Your Target Cell Line eee 12 D Protocol Testing Your Tet On Advanced Clones for Induction ssssessssesssstsrsreresrereerereerereerererreree 12 IX Developing the Double Stable Tet On Advanced Inducible Cell Line ccccceeee 13 A Functional Testing of pTRE Tight GOI in the Tet On Advanced Cell Line wo cceeeeseteeeeeeee 13 B Protocol Creating the Double Stable Tet On Advanced Inducible Cell Line wo ieee eeeeeeeneeeeee 13 C Protocol Screening Your Panel of Double Stable Tet On Advanced Inducible Cell Lines 14 X RefEren ce cccesscsssssseseseseeeceesecececeneeseseeseseceusesaeananagssusseseseseaeseeeeeeeseeeeseesesesensnsaeananesess 15 Appendix A Tet Vector Information c ccccccccccceccsssssssseeaesesseseeeecesesssenaeseesesseseseesssssssaneeseeees 16 Appendix B Titrating Antibiotics for Selecting Stable Cell Lines cccceceesseeeeeeeeeeeees 18 Appendix C Using pTRE Cycle Vectors to Rapidly Control Protein Levels c cccceeees 18 List of Figures Figure 1 Induction in the Tet On Advanced System c ecccscssssssessssssescseeseecscseeececesseseeeasscneeaeeseenanesesenaes 3 Figure 2 Tetracycline activity in bovine seraic scscscicesiscessttus shied anena eneee EEEE A EKASTA DENNEKE TENi 6 Figure 3 Establishing the Tet O
35. o PT3898 1 Version No 18 102312 www clontech com Clontech Laboratories Inc ATakara Bio Company Appendix C Tet On Advanced Inducible Gene Expression Systems User Manual Using pTRE Cycle Vectors to Rapidly Control Protein Levels C Performing a protein cycling experiment 1 Expression ON Using the double stable pTet On Advanced pTRE Cycle GOI clone as selected in Step A induce expression in the presence of both Dox and Shield1 2 Expression OFF Remove the medium containing both Dox and Shield1 Protein levels should decline very rapidly Dox and Shield1 are effectively removed by the following treatment a Wash the cells on the plate 2X with PBS Trypsinize and collect the cells wash them in suspension 1X with PBS and plate in fresh medium without Dox b After the cells have reattached to the substrate gently wash them on the plate 1X with PBS and add fresh medium without Dox 3 Expression ON Add Dox and Shield1 again In fact there are two options for control in Step C 2 e Rapid control Turn OFF by removing Shield only and retain Dox in the medium Depending on mRNA stability this may result in a more rapid subsequent ON rate since high level transcriptional activity is main tained for Step 3 e Tightest control Turn OFF by removing both Dox and Shield1 This will result in the lowest possible level of your protein of interest Contact Us For Assistance Customer Service Ordering
36. on days 1 For each clone to be tested seed an aliquot of cells in 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 2 Distribute the remaining cells among the wells of a tissue culture plate 24 96 wells so that a range of Dox concentrations 10 1000 ng ml can be tested in duplicate versus an uninduced No Dox control 3 Add Dox to the appropriate wells and incubate the cells for 48 hr 4 Harvest the cells and use an assay specific for your GOI to quantify the expression of your GOI 5 Select clones with the highest fold induction for propagation and further testing 6 Freeze stocks of each promising clone as soon as possible Protocol No PT3898 1 www clontech com Clontech Laboratories Inc Version No 102312 ATakara Bio Company 14 Tet On Advanced Inducible Gene Expression Systems User Manual X References You can access further information on Tet Systems products on our website www clontech com Clontech s Tet Systems were developed in cooperation with Dr Bujard and his colleagues at the Center for Molecular Biology in Heidelberg ZMBH and in Dr Wolfgang Hillen s laboratory at the University of Erlangen Germany Additional background information on Tet regulated gene expression systems and an extensive bibliography are available at the website maintained by TET Systems http www tetsystems com Please note that Clontech is not respons
37. s shown that testing this many clones yields a high rate of success and will prevent significant delays Your panel of Tet On Advanced cell line clones should be screened by transiently transfecting them with pTRE Tight Luc to test for high induction and low basal expression of luciferase activity When you have identified a clone that demonstrates ideal induction characteristics proceed to Section IX to develop the double stable Tet On Advanced inducible cell line Be sure to freeze aliquots of your Tet On Advanced cell line s B Protocol Pilot Testing Tet Based Induction in Target Cells While many cell backgrounds have been shown to support Tet based expression control Tet systems have not been Protocol tested in all cell lines Performing a transient expression assay with pTet On Advanced and pTRE Tight Luc pro 2 3 vides a quick indication of how well the Tet On Advanced System will work in your target cell line Transfected cells days are treated with Dox to induce expression of luciferase from pTRE Tight Luc 1 Using conditions and transfection methods appropriate for your cell line cotransfect duplicate wells of cells in 6 well plates with pTet On Advanced and pTRE Tight Luc Use several different Tet On TRE vector ra tios e g at 1 1 1 5 and 5 1 to ensure that a functional induction system is attained in the transfected cells 2 When transfection has been completed replace the transfection medium with fresh culture medium A
38. uner System Transcriptional control of your GOI is achieved with Dox while the stability of your protein is controlled with the ProteoTuner ligand Shield1 Cat No 631037 With this combination it is possible to rapidly and completely eliminate your expressed protein from cells by removing Dox and Shield 1 and have it reappear by adding Dox and Shield1 The pT RE Cycle vectors possess the following two key features e The Pigg Promoter provides tight bidirectional control of transcription mediated by Tet Advanced transactivators e The ProteoTuner destabilizing domain DD allows the stability of a protein of interest to be precisely con trolled by Shield1 Below is a brief outline of the steps needed to perform protein cycling studies We strongly recommend that you consult this Tet On Advanced Systems user manual and the ProteoTuner Systems User Manual PT4039 1 for detailed protocols describing the independent aspects of these technologies B Screening for Highly Inducible Clones 1 Create and select double stable pTet On Advanced pTRE Cycle GOI clonal cell lines see Section IX When testing these clones for induction include both Dox 0 1 1 pg ml and Shield1 50 1000 nM and compare to cells treated with Shield only 2 Select a clone that has high GOI expression in the presence of Dox Shield1 and very low basal expression with Shield1 alone This ensures that you have the tightest control of transcription Protocol N
39. ute 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 et al 1996 reported that the maximal level of luciferase expression in HeLa Tet Off cells transiently transfected with pT RE Luc is 35 fold higher than that obtained with HeLa cells transiently transfected with a plasmid expressing luciferase from the wild type CMV promoter Well characterized effector In contrast to effectors used in other systems such as ecdysone Dox is inex pensive well characterized and yields highly reproducible results Dox binds with high affinity to Tet On Advanced and is essentially nontoxic at the effective concentrations Note that Tet On Advanced Systems respond only to Dox and not to Tc Gossen amp Bujard 1995 Promoter activation is superior to repression Repression based systems require very high levels of repres sor to ensure 100 occupancy of the regulatory sites and fully shut off transcription The presence of high repressor levels also prevents rapid high level induction Yao et al 1998 For a more complete discussion of the advantages of transcription activation versus repression see Gossen et al 1993 The Tet On Advanced and Tet Off Advanced Expression Systems offer versatile expression control strat egies for transgenic mice The Tet System has become the de facto method of choice for generating reversibly in
40. ww clontech com Protocol No PT3898 1 Version No 102312 19
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