Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Development of the Mammalian Expression Vector System that can be Induced by IPTG and/or Lactose

  • Myung, Seung-Hyun (Department of Biochemistry and Molecular Biology, Chosun University School of Medicine) ;
  • Park, Junghee (Department of Biochemistry and Molecular Biology, Chosun University School of Medicine) ;
  • Han, Ji-Hye (Department of Biochemistry and Molecular Biology, Chosun University School of Medicine) ;
  • Kim, Tae-Hyoung (Department of Biochemistry and Molecular Biology, Chosun University School of Medicine)
  • Received : 2020.03.17
  • Accepted : 2020.05.03
  • Published : 2020.08.28
Download PDF
⟨ Previous Next ⟩

Abstract

Techniques used for the regulation of gene expression facilitate studies of gene function and treatment of diseases via gene therapy. Many tools have been developed for the regulation of gene expression in mammalian cells. The Lac operon system induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) is one of the employed inducible systems. IPTG mimics the molecular structure of allolactose and has a strong affinity for the corresponding repressor. IPTG is known to rapidly penetrate into mammalian cells and exhibits low toxicity. In the present study, we developed a new inducible expression system that could regulate the expression of genes in mammalian cells using IPTG. Here we confirm that unlike other vector systems based on the Lac operon, this expression system allows regulation of gene expression with lactose in the mammalian cells upon transfection. The co-treatment with IPTG and lactose could improve the regulatory efficiency of the specific target gene expression. The regulation of gene expression with lactose has several benefits. Lactose is safe in humans as compared to other chemical substances and is easily available, making this technique very cost-effective.

Keywords

References

  1. Wirth T, Parker N, Yla-Herttuala S. 2013. History of gene therapy. Gene 525: 162-169. https://doi.org/10.1016/j.gene.2013.03.137
  2. Kim TK, Eberwine JH. 2010. Mammalian cell transfection: the present and the future. Anal. Bioanal. Chem. 397: 3173-3178. https://doi.org/10.1007/s00216-010-3821-6
  3. Luten J, van Nostrum CF, De Smedt SC, Hennink WE. 2008. Biodegradable polymers as non-viral carriers for plasmid DNA delivery. J. Control Release 126: 97-110. https://doi.org/10.1016/j.jconrel.2007.10.028
  4. Xu ZL, Mizuguchi H, Ishii-Watabe A, Uchida E, Mayumi T, Hayakawa T. 2001. Optimization of transcriptional regulatory elements for constructing plasmid vectors. Gene 272: 149-156. https://doi.org/10.1016/S0378-1119(01)00550-9
  5. Sauer B, Henderson N. 1988. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc. Natl. Acad. Sci. USA 85: 5166-70. https://doi.org/10.1073/pnas.85.14.5166
  6. Sternberg N. 1981. Bacteriophage P1 site-specific recombination. III. Strand exchange during recombination at lox sites. J. Mol. Biol. 150: 603-608. https://doi.org/10.1016/0022-2836(81)90384-3
  7. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. 1998. Potent and specific genetic interference by double-stranded RNA in caenorhabditis elegans. Nature 391: 806-811. https://doi.org/10.1038/35888
  8. Saurabh S, Vidyarthi AS, Prasad D. 2014. RNA interference: concept to reality in crop improvement. Planta 239: 543-564. https://doi.org/10.1007/s00425-013-2019-5
  9. Wang QL, Li ZH. 2007. The functions of microRNAs in plants. Front. Biosci. 12: 3975-3982.
  10. Qiu S, Adema CM, Lane T. 2005. A computational study of off-target effects of RNA interference. Nucleic Acids Res. 33: 1834-1847. https://doi.org/10.1093/nar/gki324
  11. Jacob F, Monod J. 1961. Genetic regulatory mechanisms in the synthesis of proteins. J. Mol. Biol. 3: 318-356. https://doi.org/10.1016/S0022-2836(61)80072-7
  12. Brown M, Figge J, Hansen U, Wright C, Jeang KT, Khoury G, et al. 1987. lac repressor can regulate expression from a hybrid SV40 early promoter containing a lac operator in animal cells. Cell 49: 603-612. https://doi.org/10.1016/0092-8674(87)90536-8
  13. Hu MC, Davidson N. 1987. The inducible lac operator-repressor system is functional in mammalian cells. Cell 48: 555-566. https://doi.org/10.1016/0092-8674(87)90234-0
  14. Edamatsu H, Kaziro Y, Itoh H. 1997. Inducible high-level expression vector for mammalian cells, pEF-LAC carrying human elongation factor 1alpha promoter and lac operator. Gene 187: 289-294. https://doi.org/10.1016/S0378-1119(96)00768-8
  15. Scrable H. 2002. Say when: reversible control of gene expression in the mouse by lac. Semin. Cell Dev. Biol. 13: 109-119. https://doi.org/10.1016/S1084-9521(02)00017-4
  16. Deans TL, Cantor CR, Collins JJ. 2007. A tunable genetic switch based on RNAi and repressor proteins for regulating gene expression in mammalian cells. Cell 130: 363-372. https://doi.org/10.1016/j.cell.2007.05.045
  17. 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
  18. Ramos JL, Martinez-Bueno M, Molina-Henares AJ, Teran W, Watanabe K, Zhang X, et al. 2005. The TetR family of transcriptional repressors. Microbiol. Mol. Biol. Rev. 69: 326-356. https://doi.org/10.1128/MMBR.69.2.326-356.2005
  19. Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H. 1995. Transcriptional activation by tetracyclines in mammalian cells. Science 268: 1766-1769. https://doi.org/10.1126/science.7792603
  20. Baim SB, Labow MA, Levine AJ, Shenk T. 1991. A chimeric mammalian transactivator based on the lac repressor that is regulated by temperature and isopropyl beta-D-thiogalactopyranoside. Proc. Natl. Acad. Sci. USA 88: 5072-5076. https://doi.org/10.1073/pnas.88.12.5072
  21. Moullan N, Mouchiroud L, Wang X, Ryu D, Williams EG, Mottis A, et al. 2015. Tetracyclines disturb mitochondrial function across eukaryotic models: A call for caution in biomedical research. Cell Rep. 10: 1681-1691. https://doi.org/10.1016/j.celrep.2015.02.034
  22. Gilbert W, Muller-Hill B. 1966. Isolation of the lac repressor. Proc. Natl. Acad. Sci. USA 56: 1891-1898. https://doi.org/10.1073/pnas.56.6.1891
  23. Wyborski DL, Short JM. 1991. Analysis of inducers of the E. coli lac repressor system in mammalian cells and whole animals. Nucleic Acids Res. 19: 4647-4653. https://doi.org/10.1093/nar/19.17.4647
  24. Oehler S, Eismann ER, Kramer H, Muller-Hill B. 1990. The three operators of the lac operon cooperate in repression. EMBO J. 9: 973-979. https://doi.org/10.1002/j.1460-2075.1990.tb08199.x
  25. Santillan M, Mackey MC. 2008. Quantitative approaches to the study of bistability in the lac operon of Escherichia coli. J. R. Soc. Interface 5 Suppl 1: S29-39.
  26. Eismann ER, Muller-Hill B. 1990. lac repressor forms stable loops in vitro with supercoiled wild-type lac DNA containing all three natural lac operators. J. Mol. Biol. 213: 763-775. https://doi.org/10.1016/S0022-2836(05)80262-1
  27. Boshart M, Weber F, Jahn G, Dorsch-Hasler K, Fleckenstein B, Schaffner WA . 1985. A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell 41: 521-530. https://doi.org/10.1016/S0092-8674(85)80025-8
  28. Kim JH, Lee SR, Li LH, Park HJ, Park JH, Lee KY, et al. 2011. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 6: e18556. https://doi.org/10.1371/journal.pone.0018556
  29. Politi N, Pasotti L, Zucca S, Casanova M, Micoli G, Cusella De Angelis MG, et al. 2014. Half-life measurements of chemical inducers for recombinant gene expression. J. Biol. Eng. 8: 5. https://doi.org/10.1186/1754-1611-8-5
  30. Jobe A, Bourgeois S. 1973. Lac repressor-operator interaction. 8. Lactose is an anti-inducer of the lac operon. J. Mol. Biol. 75: 303-313. https://doi.org/10.1016/0022-2836(73)90023-5