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Cited by 246Gene Regulatory Network AnalysisVirus-based gene therapy researchGenomics and Chromatin Dynamics

Improved Tet-responsive promoters with minimized background expression

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Loew, Rainer, Heinz, Niels, Hampf, Mathias, Bujard, Hermann, Gossen, Manfred·2010·BMC Biotechnology
DOI 10.1186/1472-6750-10-81PMC PMC3002914
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Abstract

BACKGROUND: The performance of the tetracycline controlled transcriptional activation system (Tet system) depends critically on the choice of minimal promoters. They are indispensable to warrant low expression levels with the system turned "off". On the other hand, they must support high level of gene expression in the "on"-state. RESULTS: In this study, we systematically modified the widely used Cytomegalovirus (CMV) minimal promoter to further minimize background expression, resulting in an improved dynamic expression range. Using both plasmid-based and retroviral gene delivery, our analysis revealed that especially background expression levels could be significantly reduced when compared to previously established "standard" promoter designs. Our results also demonstrate the possibility to fine-tune expression levels in non-clonal cell populations. They also imply differences regarding the requirements for tight regulation and high level induction between transient and stable gene transfer systems. CONCLUSIONS: Until now, our understanding of mammalian transcriptional regulation including promoter architecture is limited. Nevertheless, the partly empirical modification of cis-elements as shown in this study can lead to the specific improvement of the performance of minimal promoters. The novel composite Ptet promoters introduced here will further expand the utility of the Tet system.

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  • The pTet promoter has been optimized through systematic modification, resulting in novel promoter configurations that minimized intrinsic background activity while maintaining high induced expression.

    “The pTet promoter has been optimized through systematic modification, which resulted in novel promoter configurations that minimized intrinsic background activity while maintaining high induced expression [13, 14].”

    From Tet-On Systems For Doxycycline-inducible Gene Expression (2016) · cites this paper as [13]

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Research works printed but not linked: 48

  1. Park HJ RajBhandary UL Tetracycline-regulated suppression of amber codons in mammalian cells Mol Cell Biol 1998 18 8 4418 4425 9671451 PMC109027
  2. Yoshida Y Hamada H Adenovirus-mediated inducible gene expression through tetracycline-controllable transactivator with nuclear localization signal Biochem Biophys Res Commun 1997 230 2 426 430 10.1006/bbrc.1996.5975 9016796 · DOI
  3. Wells KD Foster JA Moore K Pursel VG Wall RJ Codon optimization, genetic insulation, and an rtTA reporter improve performance of the tetracycline switch Transgenic Res 1999 8 5 371 381 10.1023/A:1008952302539 10669945 · DOI
  4. Urlinger S Baron U Thellmann M Hasan MT Bujard H Hillen W Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity Proc Natl Acad Sci USA 2000 97 14 7963 7968 10.1073/pnas.130192197 10859354 PMC16653 · DOI · Paper page
  5. Valencik ML McDonald JA Codon optimization markedly improves doxycycline regulated gene expression in the mouse heart Transgenic Res 2001 10 3 269 275 10.1023/A:1016601928465 11437283 · DOI
  6. Baron U Gossen M Bujard H Tetracycline-controlled transcription in eukaryotes: novel transactivators with graded transactivation potential Nucleic Acids Res 1997 25 14 2723 2729 10.1093/nar/25.14.2723 9207017 PMC146828 · DOI
  7. Urlinger S Helbl V Guthmann J Pook E Grimm S Hillen W The p65 domain from NF-kappaB is an efficient human activator in the tetracycline-regulatable gene expression system Gene 2000 247 1-2 103 110 10.1016/S0378-1119(00)00112-8 10773449 · DOI
  8. Das AT Zhou X Vink M Klaver B Verhoef K Marzio G Berkhout B Viral evolution as a tool to improve the tetracycline-regulated gene expression system J Biol Chem 2004 279 18 18776 18782 10.1074/jbc.M313895200 14761948 · DOI · Paper page
  9. Baron U Freundlieb S Gossen M Bujard H Co-regulation of two gene activities by tetracycline via a bidirectional promoter Nucleic Acids Res 1995 23 17 3605 3606 10.1093/nar/23.17.3605 7567477 PMC307245 · DOI
  10. Agha-Mohammadi S O'Malley M Etemad A Wang Z Xiao X Lotze MT Second-generation tetracycline-regulatable promoter: repositioned tet operator elements optimize transactivator synergy while shorter minimal promoter offers tight basal leakiness J Gene Med 2004 6 7 817 828 10.1002/jgm.566 15241789 · DOI · Paper page
  11. Pluta K Luce MJ Bao L Agha-Mohammadi S Reiser J Tight control of transgene expression by lentivirus vectors containing second-generation tetracycline-responsive promoters J Gene Med 2005 7 6 803 817 10.1002/jgm.712 15655804 · DOI
  12. Danke C Grunz X Wittmann J Schmidt A Agha-Mohammadi S Kutsch O Jack HM Hillen W Berens C Adjusting transgene expression levels in lymphocytes with a set of inducible promoters J Gene Med 2010 12 6 501 515 10.1002/jgm.1461 20527043 · DOI
  13. Hoffmann A Villalba M Journot L Spengler D A novel tetracycline-dependent expression vector with low basal expression and potent regulatory properties in various mammalian cell lines Nucleic Acids Res 1997 25 5 1078 1079 10.1093/nar/25.5.1078 9023121 PMC146524 · DOI
  14. Loew R Vigna E Lindemann D Naldini L Bujard H Retroviral vectors containing Tet-controlled bidirectional transcription units for simultaneous regulation of two gene activities J Mol Genet Med 2006 2 1 107 118 19565004 PMC2702057
  15. Weidenfeld I Gossen M Low R Kentner D Berger S Gorlich D Bartsch D Bujard H Schonig K Inducible expression of coding and inhibitory RNAs from retargetable genomic loci Nucleic Acids Res 2009 37 7 e50 10.1093/nar/gkp108 19264799 PMC2673444 · DOI
  16. Qu Z Thottassery JV Van Ginkel S Manuvakhova M Westbrook L Roland-Lazenby C Hays S Kern FG Homogeneity and long-term stability of tetracycline-regulated gene expression with low basal activity by using the rtTA2S-M2 transactivator and insulator-flanked reporter vectors Gene 2004 327 1 61 73 10.1016/j.gene.2003.10.029 14960361 · DOI
  17. Junghans RP Boone LR Skalka AM Retroviral DNA H structures: displacement-assimilation model of recombination Cell 1982 30 1 53 62 10.1016/0092-8674(82)90011-3 6181896 · DOI
  18. Junghans RP Boone LR Skalka AM Products of reverse transcription in avian retrovirus analyzed by electron microscopy J Virol 1982 43 2 544 554 6180182 PMC256158
  19. Chou S Struhl K Transcriptional activation by TFIIB mutants that are severely impaired in interaction with promoter DNA and acidic activation domains Mol Cell Biol 1997 17 12 6794 6802 9372910 PMC232535
  20. Lagrange T Kapanidis AN Tang H Reinberg D Ebright RH New core promoter element in RNA polymerase II-dependent transcription: sequence-specific DNA binding by transcription factor IIB Genes Dev 1998 12 1 34 44 10.1101/gad.12.1.34 9420329 PMC316406 · DOI
  21. Evans R Fairley JA Roberts SG Activator-mediated disruption of sequence-specific DNA contacts by the general transcription factor TFIIB Genes Dev 2001 15 22 2945 2949 10.1101/gad.206901 11711430 PMC312826 · DOI
  22. Gupta R Emili A Pan G Xiao H Shales M Greenblatt J Ingles CJ Characterization of the interaction between the acidic activation domain of VP16 and the RNA polymerase II initiation factor TFIIB Nucleic Acids Res 1996 24 12 2324 2330 10.1093/nar/24.12.2324 8710503 PMC145954 · DOI
  23. Hayashi F Ishima R Liu D Tong KI Kim S Reinberg D Bagby S Ikura M Human general transcription factor TFIIB: conformational variability and interaction with VP16 activation domain Biochemistry 1998 37 22 7941 7951 10.1021/bi9801098 9609687 · DOI
  24. Lee M Struhl K Mutations on the DNA-binding surface of TATA-binding protein can specifically impair the response to acidic activators in vivo Mol Cell Biol 1995 15 10 5461 5469 7565697 PMC230796
  25. Chen Z Manley JL Core promoter elements and TAFs contribute to the diversity of transcriptional activation in vertebrates Mol Cell Biol 2003 23 20 7350 7362 10.1128/MCB.23.20.7350-7362.2003 14517303 PMC230314 · DOI
  26. Ghazal P Nelson JA Enhancement of RNA polymerase II initiation complexes by a novel DNA control domain downstream from the cap site of the cytomegalovirus major immediate-early promoter J Virol 1991 65 5 2299 2307 1850012 PMC240579
  27. Macias MP Huang L Lashmit PE Stinski MF Cellular or viral protein binding to a cytomegalovirus promoter transcription initiation site: effects on transcription J Virol 1996 70 6 3628 3635 8648697 PMC190238
  28. Bink HH Schirawski J Haenni AL Pleij CW The 5'-proximal hairpin of turnip yellow mosaic virus RNA: its role in translation and encapsidation J Virol 2003 77 13 7452 7458 10.1128/JVI.77.13.7452-7458.2003 12805444 PMC164824 · DOI
  29. Hellendoorn K Verlaan PW Pleij CW A functional role for the conserved protonatable hairpins in the 5' untranslated region of turnip yellow mosaic virus RNA J Virol 1997 71 11 8774 8779 9343237 PMC192343
  30. Bonin AL Gossen M Bujard H Photinus pyralis luciferase: vectors that contain a modified luc coding sequence allowing convenient transfer into other systems Gene 1994 141 1 75 77 10.1016/0378-1119(94)90130-9 8163178 · DOI
  31. Fehse B Kustikova OS Bubenheim M Baum C Pois(s)on--it's a question of dose Gene Ther 2004 11 11 879 881 10.1038/sj.gt.3302270 15057265 · DOI
  32. Austin CD De Maziere AM Pisacane PI van Dijk SM Eigenbrot C Sliwkowski MX Klumperman J Scheller RH Endocytosis and sorting of ErbB2 and the site of action of cancer therapeutics trastuzumab and geldanamycin Mol Biol Cell 2004 15 12 5268 5282 10.1091/mbc.E04-07-0591 15385631 PMC532009 · DOI
  33. Carlsson J Nordgren H Sjostrom J Wester K Villman K Bengtsson NO Ostenstad B Lundqvist H Blomqvist C HER2 expression in breast cancer primary tumours and corresponding metastases. Original data and literature review Br J Cancer 2004 90 12 2344 2348 15150568 PMC2409528
  34. Wu X Li Y Crise B Burgess SM Transcription start regions in the human genome are favored targets for MLV integration Science 2003 300 5626 1749 1751 10.1126/science.1083413 12805549 · DOI · Paper page
  35. Deng W Roberts SG TFIIB and the regulation of transcription by RNA polymerase II Chromosoma 2007 116 5 417 429 10.1007/s00412-007-0113-9 17593382 · DOI
  36. Wu WH Hampsey M An activation-specific role for transcription factor TFIIB in vivo Proc Natl Acad Sci USA 1999 96 6 2764 2769 10.1073/pnas.96.6.2764 10077585 PMC15843 · DOI
  37. Pesole G Mignone F Gissi C Grillo G Licciulli F Liuni S Structural and functional features of eukaryotic mRNA untranslated regions Gene 2001 276 1-2 73 81 10.1016/S0378-1119(01)00674-6 11591473 · DOI
  38. Soneoka Y Cannon PM Ramsdale EE Griffiths JC Romano G Kingsman SM Kingsman AJ A transient three-plasmid expression system for the production of high titer retroviral vectors Nucleic Acids Res 1995 23 4 628 633 10.1093/nar/23.4.628 7899083 PMC306730 · DOI
  39. Pietschmann T Heinkelein M Heldmann M Zentgraf H Rethwilm A Lindemann D Foamy virus capsids require the cognate envelope protein for particle export J Virol 1999 73 4 2613 2621 10074106 PMC104016
  40. Hampf M Gossen M A protocol for combined Photinus and Renilla luciferase quantification compatible with protein assays Anal Biochem 2006 356 1 94 99 10.1016/j.ab.2006.04.046 16750160 · DOI
  41. Loew R The use of retroviral vectors for tet-regulated gene expression in cell populations Methods Mol Biol 2009 506 221 242 full_text 19110630
  42. Bradford MM A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 1976 72 248 254 10.1016/0003-2697(76)90527-3 942051 · DOI · Paper page
  43. Leclerc GM Boockfor FR Faught WJ Frawley LS Development of a destabilized firefly luciferase enzyme for measurement of gene expression Biotechniques 2000 29 3 590 591 594-596, 598 passim 10997273
  44. Wahlers A Schwieger M Li Z Meier-Tackmann D Lindemann C Eckert HG von Laer D Baum C Influence of multiplicity of infection and protein stability on retroviral vector-mediated gene expression in hematopoietic cells Gene Ther 2001 8 6 477 486 10.1038/sj.gt.3301426 11313827 · DOI
  45. Wang Y Wang G O'Kane DJ Szalay AA A study of protein-protein interactions in living cells using luminescence resonance energy transfer (LRET) from Renilla luciferase to Aequorea GFP Mol Gen Genet 2001 264 5 578 587 10.1007/s004380000322 11212912 · DOI
  46. Heim R Cubitt AB Tsien RY Improved green fluorescence Nature 1995 373 6516 663 664 10.1038/373663b0 7854443 · DOI
  47. Ausubel FM Brent R Kingston RE Moore DD Seidman JG Smith JA Struhl K Current protocols in molecular biology 2001 1 John Wiley&Sons, Inc
  48. Zufferey R Donello JE Trono D Hope TJ Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors J Virol 1999 73 4 2886 2892 10074136 PMC104046

Cited by

Sorted by citations

Authors
Loew, R., Heinz, N., Hampf, M., Bujard, H., & Gossen, M.
Affiliations
  • Medizinische Hochschule Hannover (DE)
  • Max Delbrück Center (DE)
  • Heidelberg University (DE)
Corresponding author
Loew, Rainer
ORCID
Gossen, Manfred
Venue
BMC Biotechnology
Publisher
Springer Science and Business Media LLC
Published
2010
Volume
10
Issue
1
Pages
81
ISSN
1472-6750 (ISSN-L)
DOI
10.1186/1472-6750-10-81
PMC
PMC3002914
OpenAlex
W2086322167
Cited by
246
References
4
Citations by year
2026: 19 · 2025: 17 · 2024: 23 · 2023: 24 · 2022: 20 · 2021: 18 · 2020: 27 · 2019: 15 · 2018: 14 · 2017: 10 · 2016: 15 · 2015: 13 · 2014: 5 · 2013: 15 · 2012: 6
FWCI
3.39
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • BMC Biotechnology (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • MDC Repository (Max-Delbrueck-Center for Molecular Medicine) (acceptedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Bundesministerium für Bildung und Forschung
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
Keywords
minimal promoter, cytomegalovirus promoter, promoter engineering, cis-regulatory elements, retroviral gene transfer, stable gene transfer, gene expression regulation
MeSH terms
Cell Line, Gene Expression Regulation, Genetic Vectors, Humans, Promoter Regions, Genetic, Tetracycline, Transduction, Genetic, Transgenes

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