Reproductive and Developmental Biology

The Korean Society of Animal Reproduction (한국동물번식학회)
권호 표지
DOI QR코드

DOI QR Code

Examination of Improved Tetracycline Inducible Gene Expression System In Vitro

새로운 Tetracycline 유도적 유전자 발현 System의 In Vitro 검정

  • Kwon, Mo Sun (Department of Physiology, Catholic University of Daegu School of Medicine) ;
  • Kim, Teoan (Department of Physiology, Catholic University of Daegu School of Medicine) ;
  • Koo, Bon Chul (Department of Physiology, Catholic University of Daegu School of Medicine)
  • 권모선 (대구가톨릭대학교 의과대학 생리학교실) ;
  • 김태완 (대구가톨릭대학교 의과대학 생리학교실) ;
  • 구본철 (대구가톨릭대학교 의과대학 생리학교실)
  • Received : 2013.09.06
  • Accepted : 2013.09.10
  • Published : 2013.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

Until recently the most popular tetracycline-inducible gene expression system has been the one developed by Gossen and Bujard. In this study, we tested the latest version of same system and the results are summarized as follows: Compared with previous one, the difference of new system are minor changes of nucleotide sequences in transactivator and tetracycline response element (TRE) regions. Sensitivity to the doxycycline (a tetracycline derivative) was improved. Leakiness of GFP marker gene expression in non-inducible condition was significantly decreased. Higher expression of the marker gene was observed when the cells were fed with doxycycline-containing medium. Optimal insertion site of woodchuck posttranscriptional regulatory element (WPRE) sequence which was known to increase gene expression was different depending on the origin of cells. In chicken embryonic fibroblast, location of WPRE sequence at 3' end of TRE resulted in the highest GFP expression. In bovine embryonic fibroblasts, 3' end of transactivator was the best site for the GFP expression.

Keywords

References

  1. Ahfeldt T, Schinzel RT, Lee YK, Hendrickson D, Kaplan A, Lum DH, Camahort R, Xia F, Shay J, Rhee EP, Clish CB, Deo RC, Shen T, Lau FH, Cowley A, Mowrer G, Al-Siddiqi H, Nahrendorf M, Musunuru K, Gerszten RE, Rinn JL, Cowan CA (2012): Programming human pluripotent stem cells into white and brown adipocytes. Nat Cell Biol 14:209- 219. https://doi.org/10.1038/ncb2411
  2. Backman CM, Zhang Y, Hoffer BJ, Tomac AC (2004): Tetracycline-inducible expression systems for the generation of transgenic animals: a comparison of various inducible systems carried in a single vector. J Neurosci Methods 139:257-262. https://doi.org/10.1016/j.jneumeth.2004.05.012
  3. Bohl D, Naffakh N, Heard JM (1997): Long-term control of erythropoietin secretion by doxycycline in mice with engineered primary myoblasts. Nature Med 3:299-305. https://doi.org/10.1038/nm0397-299
  4. 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. https://doi.org/10.1073/pnas.89.12.5547
  5. Gossen M, Freundlieb S, Bender G, Müller G, Hillen W, Bujard H (1995): Transcriptional activation by tetracyclines in mammalian cells. Science 268: 1766-1769. https://doi.org/10.1126/science.7792603
  6. Kim MJ, Oh HJ, Park JE, Kim GA, Hong SG, Jang G, Kwon MS, Koo BC, Kim T, Kang SK, Ra JC, Ko C, Lee BC (2011): Generation of transgenic dogs that conditionally express green fluorescent protein. Genesis 49:472-478. https://doi.org/10.1002/dvg.20737
  7. Koo BC, Kwon MS, Kim T (2005): Regulation of GFP expression using the tetracycline inducible retroviral vector system. Reprod Dev Biol 29:57-62.
  8. Koo BC, Kwon MS, Kim T (2009): Construction of improved tetracycline-inducible expression system for the effective regulation of transgene expression. Reprod Dev Biol 33:63-69.
  9. Lamartina S, Roscilli G, Rinaudo CD, Sporeno E, Silvi L, Hillen W, Bujard H, Cortese R, Ciliberto G, Toniatti C (2002): Stringent control of gene expression in vivo by using novel doxycycline-dependent trans-activators. Hum Gene Ther 13:199- 210. https://doi.org/10.1089/10430340252769734
  10. Li Z, Huang X, Zhan H, Zeng Z, Li C, Spitsbergen JM, Meierjohann S, Schartl M, Gong Z (2012): Inducible and repressable oncogene-addicted hepatocellular carcinoma in Tet-on xmrk transgenic zebrafish. J Hepatol 56:419-425.
  11. Low R, Heinz N, Hampf M, Bujard H, Gossen M (2010): Improved Tet-responsive promoters with minimized background expression. BMC Biotechnology 2010, 10:81. https://doi.org/10.1186/1472-6750-10-81
  12. Mayford M, Bach ME, Huang YY, Wang L, Hawkins RD, Kandel ER (1996): Control of mamary formation through regulated-expression of a CaMK transgene. Science 274:1678-1683. https://doi.org/10.1126/science.274.5293.1678
  13. Meerbrey KL, Hu G, Kessler JD, Roarty K, Li MZ, Fang JE, Herschkowitz JI, Burrows AE, Ciccia A, Sun T, Schmitt EM, Bernardi RJ, Fu X, Bland CS, Cooper TA, Schiff R, Rosen JM, Westbrook TF, Elledge SJ (2011): The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo. Proc Natl Acad Sci USA 108:3665-3670. https://doi.org/10.1073/pnas.1019736108
  14. Ryding ADS, Sharp MGF, Mullins JJ (2001): Conditional transgenic technologies. J Endocrinol 171:1-14. https://doi.org/10.1677/joe.0.1710001
  15. Sun T, Aceto N, Meerbrey KL, Kessler JD, Zhou C, Migliaccio I, Nguyen DX, Pavlova NN, Botero M, Huang J, Bernardi RJ, Schmitt E, Hu G, Li MZ, Dephoure N, Gygi SP, Rao M, Creighton CJ, Hilsenbeck SG, Shaw CA, Muzny D, Gibbs RA, Wheeler DA, Osborne CK, Schiff R, Bentires-Alj M, Elledge SJ, Westbrook TF (2011): Activation of multiple proto-oncogenic tyrosine kinases in breast cancer via loss of the PTPN12 phosphatase. Cell 144:703- 718. https://doi.org/10.1016/j.cell.2011.02.003
  16. Thorel F, Nepote V, Avril I, Kohno K, Desgraz R, Chera S, Herrera PL (2010): Conversion of adult pancreatic alpha-cells to beta-cells after extreme beta- cell loss. Nature 464:1149-1154. https://doi.org/10.1038/nature08894
  17. Traykova-Brauch M, Schonig K, Greiner O, Miloud T, Jauch A, Bode M, Felsher DW, Glick AB, Kwiatkowski DJ, Bujard H, Horst J, von Knebel Doeberitz M, Niggli FK, Kriz W, Gröne HJ, Koesters R (2008): An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice. Nat Med 14:979-984. https://doi.org/10.1038/nm.1865
  18. 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. https://doi.org/10.1073/pnas.130192197
  19. Wang J, Theunissen TW, Orkin SH (2007): Site-directed, virus-free, and inducible RNAi in embryo- nic stem cells. Proc Natl Acad Sci USA 104:20850- 20855. https://doi.org/10.1073/pnas.0710565105
  20. Woltjen K, Michael IP, Mohseni P, Desai R, Mileikovsky M, Hamalainen R, Cowling R, Wang W, Liu P, Gertsenstein M, Kaji K, Sung HK, Nagy A (2009): piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 458: 766-770. (Received: 6 September 2013/ Accepted: 10 September 2013) https://doi.org/10.1038/nature07863