Skip to main content
SpringerLink
Log in

Development of a BAC vector for integration-independent and tight regulation of transgenes in rodents via the Tet system

  • Technical Report
  • Published:
Transgenic Research Aims and scope Submit manuscript

Abstract

The establishment of functional transgenic mouse lines is often limited by problems caused by integration site effects on the expression construct. Similarly, tetracycline (Tet) controlled transcription units most commonly used for conditional transgene expression in mice are strongly influenced by their genomic surrounding. Using bacterial artificial chromosome (BAC) technology in constitutive expression systems, it has been shown that integration site effects resulting in unwanted expression patterns can be largely eliminated. Here we describe a strategy to minimize unfavourable integration effects on conditional expression constructs based on a 75 kb genomic BAC fragment. This fragment was derived from a transgenic mouse line, termed LC-1, which carries the Tet-inducible genes luciferase and cre (Schönig et al. 2002). Animals of this mouse line have previously been shown to exhibit optimal expression properties in terms of tightness in the off state and the absolute level of induction, when mated to appropriate transactivator expressing mice. Here we report the cloning and identification of the transgenic LC-1 integration site which was subsequently inserted into a bacterial artificial chromosome. We demonstrate that this vector facilitates the efficient generation of transgenic mouse and rat lines, where the Tet-controlled expression unit is shielded from perturbations caused by the integration site.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  • Baer A, Bode J (2001) Coping with kinetic and thermodynamic barriers: RMCE, an efficient strategy for the targeted integration of transgenes. Curr Opin Biotechnol 12:473–480

    Article  PubMed  CAS  Google Scholar 

  • Baron U, Bujard H (2000) Tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances. Methods Enzymol 327:401–421

    Article  PubMed  CAS  Google Scholar 

  • Baron U, Freundlieb S, Gossen M, Bujard H (1995) Co-regulation of two gene activities by tetracycline via a bidirectional promoter. Nucleic Acids Res 23:3605–3606

    Article  PubMed  CAS  Google Scholar 

  • Beard C, Hochedlinger K, Plath K, Wutz A, Jaenisch R (2006) Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells. Genesis 44:23–28

    Article  PubMed  CAS  Google Scholar 

  • Birren B, Lai E (1993) Pulsed field electrophoresis: a practical guide. Academic Press, San Diego

    Google Scholar 

  • Blomfield IC, Vaughn V, Rest RF, Eisenstein BI (1991) Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon. Mol Microbiol 5:1447–1457

    Article  PubMed  CAS  Google Scholar 

  • Bockamp E, Antunes C, Maringer M, Heck R, Presser K, Beilke S, Ohngemach S, Alt R, Cross M, Sprengel R, Hartwig U, Kaina B, Schmitt S, Eshkind L (2006) Tetracycline-controlled transgenic targeting from the SCL locus directs conditional expression to erythrocytes, megakaryocytes, granulocytes, and c-kit-expressing lineage-negative hematopoietic cells. Blood 108:1533–1541

    Article  PubMed  CAS  Google Scholar 

  • Bode J, Schlake T, Iber M, Schubeler D, Seibler J, Snezhkov E, Nikolaev L (2000) The transgeneticist’s toolbox: novel methods for the targeted modification of eukaryotic genomes. Biol Chem 381:801–813

    Article  PubMed  CAS  Google Scholar 

  • Bonnerot C, Grimber G, Briand P, Nicolas JF (1990) Patterns of expression of position-dependent integrated transgenes in mouse embryo. Proc Natl Acad Sci USA 87:6331–6335

    Article  PubMed  CAS  Google Scholar 

  • Boross P, Breukel C, van Loo PF, van der Kaa J, Claassens JW, Bujard H, Schonig K, Verbeek JS (2009) Highly B lymphocyte-specific tamoxifen inducible transgene expression of CreER(T2) by using the LC-1 locus BAC vector. Genesis 47(11):729–735

    Article  PubMed  CAS  Google Scholar 

  • Buehr M, Meek S, Blair K, Yang J, Ure J, Silva J, McLay R, Hall J, Ying QL, Smith A (2008) Capture of authentic embryonic stem cells from rat blastocysts. Cell 135:1287–1298

    Article  PubMed  CAS  Google Scholar 

  • Burgin KE, Waxham MN, Rickling S, Westgate SA, Mobley WC, Kelly PT (1990) In situ hybridization histochemistry of Ca2 +/calmodulin-dependent protein kinase in developing rat brain. J Neurosci 10:1788–1798

    PubMed  CAS  Google Scholar 

  • Casanova E, Fehsenfeld S, Mantamadiotis T, Lemberger T, Greiner E, Stewart AF, Schutz G (2001) A CamKIIalpha iCre BAC allows brain-specific gene inactivation. Genesis 31:37–42

    Article  PubMed  CAS  Google Scholar 

  • Clark AJ, Bissinger P, Bullock DW, Damak S, Wallace R, Whitelaw CB, Yull F (1994) Chromosomal position effects and the modulation of transgene expression. Reprod Fertil Dev 6:589–598

    Article  PubMed  CAS  Google Scholar 

  • Cohen B, Ziv K, Plaks V, Israely T, Kalchenko V, Harmelin A, Benjamin LE, Neeman M (2007) MRI detection of transcriptional regulation of gene expression in transgenic mice. Nat Med 13:498–503

    Article  PubMed  CAS  Google Scholar 

  • Gallagher AR, Schonig K, Brown N, Bujard H, Witzgall R (2003) Use of the tetracycline system for inducible protein synthesis in the kidney. J Am Soc Nephrol 14:2042–2051

    Article  PubMed  CAS  Google Scholar 

  • Giraldo P, Montoliu L (2001) Size matters: use of YACs, BACs and PACs in transgenic animals. Transgenic Res 10:83–103

    Article  PubMed  CAS  Google Scholar 

  • Gossen M, Bujard H (1992) Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 89:5547–5551

    Article  PubMed  CAS  Google Scholar 

  • Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H (1995) Transcriptional activation by tetracyclines in mammalian cells. Science 268:1766–1769

    Article  PubMed  CAS  Google Scholar 

  • Hampf M, Gossen M (2007) Promoter crosstalk effects on gene expression. J Mol Biol 365:911–920

    Article  PubMed  CAS  Google Scholar 

  • Hasan MT, Friedrich RW, Euler T, Larkum ME, Giese G, Both M, Duebel J, Waters J, Bujard H, Griesbeck O, Tsien RY, Nagai T, Miyawaki A, Denk W (2004) Functional fluorescent Ca2 + indicator proteins in transgenic mice under TET control. PLoS Biol 2:e163

    Article  PubMed  Google Scholar 

  • Herms J, Zurmohle U, Brysch W, Schlingensiepen KH (1993) Ca2 +/calmodulin protein kinase and protein kinase C expression during development of rat hippocampus. Dev Neurosci 15:410–416

    Article  PubMed  CAS  Google Scholar 

  • Higgins DF, Biju MP, Akai Y, Wutz A, Johnson RS, Haase VH (2004) Hypoxic induction of Ctgf is directly mediated by Hif-1. Am J Physiol Renal Physiol 287:F1223–F1232

    Article  PubMed  CAS  Google Scholar 

  • Hogan H, Beddington R, Constantini F, Lacy E (1994) Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  • Izumi M, Gilbert DM (1999) Homogeneous tetracycline-regulatable gene expression in mammalian fibroblasts. J Cell Biochem 76:280–289

    Article  PubMed  CAS  Google Scholar 

  • Kalos M, Fournier RE (1995) Position-independent transgene expression mediated by boundary elements from the apolipoprotein B chromatin domain. Mol Cell Biol 15:198–207

    PubMed  CAS  Google Scholar 

  • Kim UJ, Birren BW, Slepak T, Mancino V, Boysen C, Kang HL, Simon MI, Shizuya H (1996) Construction and characterization of a human bacterial artificial chromosome library. Genomics 34:213–218

    Article  PubMed  CAS  Google Scholar 

  • Kistner A, Gossen M, Zimmermann F, Jerecic J, Ullmer C, Lubbert H, Bujard H (1996) Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice. Proc Natl Acad Sci USA 93:10933–10938

    Article  PubMed  CAS  Google Scholar 

  • Krestel HE, Shimshek DR, Jensen V, Nevian T, Kim J, Geng Y, Bast T, Depaulis A, Schönig K, Schwenk F, Bujard H, Hvalby O, Sprengel R, Seeburg PH (2004) A genetic switch for epilepsy in adult mice. J Neurosci 24:10568–10578

    Article  PubMed  CAS  Google Scholar 

  • Lavon I, Goldberg I, Amit S, Landsman L, Jung S, Tsuberi BZ, Barshack I, Kopolovic J, Galun E, Bujard H, Ben-Neriah Y (2000) High susceptibility to bacterial infection, but no liver dysfunction, in mice compromised for hepatocyte NF-kappaB activation. Nat Med 6:573–577

    Article  PubMed  CAS  Google Scholar 

  • Lee EC, Yu D, Martinez de Velasco J, Tessarollo L, Swing DA, Court DL, Jenkins NA, Copeland NG (2001) A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73:56–65

    Article  PubMed  CAS  Google Scholar 

  • Lemberger T, Parlato R, Dassesse D, Westphal M, Casanova E, Turiault M, Tronche F, Schiffmann SN, Schutz G (2007) Expression of Cre recombinase in dopaminoceptive neurons. BMC Neurosci 8:4

    Article  PubMed  Google Scholar 

  • Lewandoski M (2001) Conditional control of gene expression in the mouse. Nat Rev Genet 2:743–755

    Article  PubMed  CAS  Google Scholar 

  • Li P, Tong C, Mehrian-Shai R, Jia L, Wu N, Yan Y, Maxson RE, Schulze EN, Song H, Hsieh CL, Pera MF, Ying QL (2008) Germline competent embryonic stem cells derived from rat blastocysts. Cell 135:1299–1310

    Article  PubMed  CAS  Google Scholar 

  • Loew R, Vigna E, Lindemann D, Naldini L, Bujard H (2006) Retroviral vectors containing Tet-controlled bidirectional transcription units for simultaneous regulation of two gene activities. J Mol Genet Med 2:107–118

    PubMed  Google Scholar 

  • Lutz R, Bujard H (1997) Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1–I2 regulatory elements. Nucleic Acids Res 25:1203–1210

    Article  PubMed  CAS  Google Scholar 

  • Masui S, Shimosato D, Toyooka Y, Yagi R, Takahashi K, Niwa H (2005) An efficient system to establish multiple embryonic stem cell lines carrying an inducible expression unit. Nucleic Acids Res 33:e43

    Article  PubMed  Google Scholar 

  • Mayford M, Bach ME, Huang YY, Wang L, Hawkins RD, Kandel ER (1996) Control of memory formation through regulated expression of a CaMKII transgene. Science 274:1678–1683

    Article  PubMed  CAS  Google Scholar 

  • Muyrers JP, Zhang Y, Benes V, Testa G, Rientjes JM, Stewart AF (2004) ET recombination: DNA engineering using homologous recombination in E. coli. Methods Mol Biol 256:107–121

    PubMed  CAS  Google Scholar 

  • Namciu SJ, Blochlinger KB, Fournier RE (1998) Human matrix attachment regions insulate transgene expression from chromosomal position effects in Drosophila melanogaster. Mol Cell Biol 18:2382–2391

    PubMed  CAS  Google Scholar 

  • Nielsen LB, McCormick SP, Young SG (1999) A new approach for studying gene regulation by distant DNA elements in transgenic mice. Scand J Clin Lab Invest Suppl 229:33–39

    Article  PubMed  CAS  Google Scholar 

  • Palais G, Nguyen Dinh Cat A, Friedman H, Panek-Huet N, Millet A, Tronche F, Gellen B, Mercadier JJ, Peterson A, Jaisser F (2009) Targeted transgenesis at the HPRT locus: an efficient strategy to achieve tightly controlled in vivo conditional expression with the tet system. Physiol Genom 37:140–146

    Article  CAS  Google Scholar 

  • Rodriguez I, Del Punta K, Rothman A, Ishii T, Mombaerts P (2002) Multiple new and isolated families within the mouse superfamily of V1r vomeronasal receptors. Nat Neurosci 5:134–140

    Article  PubMed  CAS  Google Scholar 

  • Schönig K, Bujard H (2003) Generating conditional mouse mutants via tetracycline-controlled gene expression. Methods Mol Biol 209:69–104

    PubMed  Google Scholar 

  • Schönig K, Schwenk F, Rajewsky K, Bujard H (2002) Stringent doxycycline dependent control of CRE recombinase in vivo. Nucleic Acids Res 30:e134

    Article  PubMed  Google Scholar 

  • Schwenk F, Baron U, Rajewsky K (1995) A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. Nucleic Acids Res 23:5080–5081

    Article  PubMed  CAS  Google Scholar 

  • Seibler J, Bode J (1997) Double-reciprocal crossover mediated by FLP-recombinase: a concept and an assay. Biochemistry 36:1740–1747

    Article  PubMed  CAS  Google Scholar 

  • Shizuya H, Birren B, Kim UJ, Mancino V, Slepak T, Tachiiri Y, Simon M (1992) Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc Natl Acad Sci USA 89:8794–8797

    Article  PubMed  CAS  Google Scholar 

  • Testa G, Vintersten K, Zhang Y, Benes V, Muyrers JP, Stewart AF (2004) BAC engineering for the generation of ES cell-targeting constructs and mouse transgenes. Methods Mol Biol 256:123–139

    PubMed  CAS  Google Scholar 

  • Urlinger S, Baron U, Thellmann M, Hasan MT, Bujard H, Hillen W (2000) Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc Natl Acad Sci USA 97:7963–7968

    Article  PubMed  CAS  Google Scholar 

  • Utomo AR, Nikitin AY, Lee WH (1999) Temporal, spatial, and cell type-specific control of Cre-mediated DNA recombination in transgenic mice. Nat Biotechnol 17:1091–1096

    Article  PubMed  CAS  Google Scholar 

  • Van Keuren ML, Gavrilina GB, Filipiak WE, Zeidler MG, Saunders TL (2009) Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression outcomes. Transgenic Res 18:769–785

    Article  PubMed  CAS  Google Scholar 

  • Wang ML, Huang L, Bongard-Pierce DK, Belmonte S, Zachgo EA, Morris JW, Dolan M, Goodman HM (1997) Construction of an approximately 2 Mb contig in the region around 80 cM of Arabidopsis thaliana chromosome 2. Plant J 12:711–730

    Article  PubMed  CAS  Google Scholar 

  • Weidenfeld I, Gossen M, Low R, Kentner D, Berger S, Görlich D, Bartsch D, Bujard H, Schönig K (2009) Inducible expression of coding and inhibitory RNAs from retargetable genomic loci. Nucleic Acids Res 37:e50

    Article  PubMed  Google Scholar 

  • Wells KD, Foster JA, Moore K, Pursel VG, Wall RJ (1999) Codon optimization, genetic insulation, and an rtTA reporter improve performance of the tetracycline switch. Transgenic Res 8:371–381

    Article  PubMed  CAS  Google Scholar 

  • Williams A, Harker N, Ktistaki E, Veiga-Fernandes H, Roderick K, Tolaini M, Norton T, Williams K, Kioussis D (2008) Position effect variegation and imprinting of transgenes in lymphocytes. Nucleic Acids Res 36:2320–2329

    Article  PubMed  CAS  Google Scholar 

  • Wilson C, Bellen HJ, Gehring WJ (1990) Position effects on eukaryotic gene expression. Annu Rev Cell Biol 6:679–714

    Article  PubMed  CAS  Google Scholar 

  • Yu D, Ellis HM, Lee EC, Jenkins NA, Copeland NG, Court DL (2000) An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci USA 97:5978–5983

    Article  PubMed  CAS  Google Scholar 

  • Yu J, Muller H, Hehn S, Koschmieder S, Schonig K, Berdel WE, Serve H, Muller-Tidow C (2009) Construction and application of an inducible system for homogenous expression levels in bulk cell lines. PLoS One 4:e6445

    Article  PubMed  Google Scholar 

  • Zahn-Zabal M, Kobr M, Girod PA, Imhof M, Chatellard P, de Jesus M, Wurm F, Mermod N (2001) Development of stable cell lines for production or regulated expression using matrix attachment regions. J Biotechnol 87:29–42

    Article  PubMed  CAS  Google Scholar 

  • Zeng H, Horie K, Madisen L, Pavlova MN, Gragerova G, Rohde AD, Schimpf BA, Liang Y, Ojala E, Kramer F, Roth P, Slobodskaya O, Dolka I, Southon EA, Tessarollo L, Bornfeldt KE, Gragerov A, Pavlakis GN, Gaitanaris GA (2008) An inducible and reversible mouse genetic rescue system. PLoS Genet 4:e1000069

    Article  PubMed  Google Scholar 

  • Zhang Y, Buchholz F, Muyrers JP, Stewart AF (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet 20:123–128

    Article  PubMed  CAS  Google Scholar 

  • Zhu Z, Ma B, Homer RJ, Zheng T, Elias JA (2001) Use of the tetracycline-controlled transcriptional silencer (tTS) to eliminate transgene leak in inducible overexpression transgenic mice. J Biol Chem 276:25222–25229

    Article  PubMed  CAS  Google Scholar 

  • Zhu P, Aller MI, Baron U, Cambridge S, Bausen M, Herb J, Sawinski J, Cetin A, Osten P, Nelson ML, Kugler S, Seeburg PH, Sprengel R, Hasan MT (2007) Silencing and un-silencing of tetracycline-controlled genes in neurons. PLoS One 2:e533

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Frank Zimmermann, Sascha Dlugosz and Ariana Frömmig for DNA microinjections, Christiane Schirra-Müller, Lena Wendler and Brigitte Pesold for technical assistance, the team of the animal care facilities at the University of Heidelberg and the Central Institute of Mental Health for assistance in animal handling. We also thank Udo Baron, Tillmann Weber and Hernando Del Portillio for expert advice and the support in large insert cloning strategies. This work was supported by Deutsche Forschungsgemeinschaft (DFG), SFB 636A1 and Bundesministerium für Bildung und Forschung (BMBF), NGFNplus.

Author information

Author notes

  1. Tina Baldinger

    Present address: Klinik und Poliklink für Neurologie, Charité -Universitätsmedizin, Berlin, Germany

Authors and Affiliations

  1. Department of Molecular Biology, Central Institute of Metal Health and Heidelberg University, Medical Faculty Mannheim, J5, 68159, Mannheim, Germany

    Kai Schönig & Dusan Bartsch

  2. Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), INF 282, 69120, Heidelberg, Germany

    David Kentner & Hermann Bujard

  3. Berlin-Brandenburg Center for Regenerative Therapies, Föhrer Straße 15, 13353, Berlin, Germany

    Manfred Gossen

  4. Department of Entomology, The Pennsylvania State University, 501 ASI Building, University Park, PA, 16802, USA

    Jun Miao

  5. Deutsches Ressourcenzentrum für Genomforschung, RZPD, Berlin, Germany

    Katrin Welzel & Andreas Vente

Authors
  1. Kai Schönig
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David Kentner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Manfred Gossen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tina Baldinger
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jun Miao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Katrin Welzel
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Andreas Vente
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Dusan Bartsch
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hermann Bujard
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kai Schönig.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schönig, K., Kentner, D., Gossen, M. et al. Development of a BAC vector for integration-independent and tight regulation of transgenes in rodents via the Tet system. Transgenic Res 20, 709–720 (2011). https://doi.org/10.1007/s11248-010-9427-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11248-010-9427-0

Keywords

Search

Navigation