Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
권호 표지
DOI QR코드

DOI QR Code

Cell Type-Specific and Inducible PTEN Gene Silencing by a Tetracycline Transcriptional Activator-Regulated Short Hairpin RNA

  • Wang, Shan (State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University) ;
  • Wang, Ting (State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University) ;
  • Wang, Tao (State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University) ;
  • Jia, Lintao (State Key Laboratory of Cancer Biology, Department of Biochemistry and Molecular Biology, Fourth Military Medical University)
  • Received : 2015.05.22
  • Accepted : 2015.07.27
  • Published : 2015.11.30
Download PDF
⟨ Previous Next ⟩

Abstract

Inducible and reversible gene silencing in desired types of cells is instrumental for deciphering gene functions using cultured cells or in vivo models. However, efficient conditional gene knockdown systems remain to be established. Here, we report the generation of an inducible expression system for short hairpin RNA (shRNA) targeted to PTEN, a well-documented dual-specificity phosphatase involved in tumor suppression and ontogenesis. Upon induction by doxycycline (DOX), the reverse tetracycline transcriptional activator (rtTA) switched on the concomitant expression of GFP and a miR-30 precursor, the subsequent processing of which released the embedded PTEN-targeted shRNA. The efficacy and reversibility of PTEN knockdown by this construct was validated in normal and neoplastic cells, in which PTEN deficiency resulted in accelerated cell proliferation, suppressed apoptosis, and increased invasiveness. Transgenic mice harboring the conditional shRNA-expression cassette were obtained; GFP expression and concurrent PTEN silencing were observed upon ectopic expression of rtTA and induction with Dox. Therefore, this study provides novel tools for the precise dissection of PTEN functions and the generation of PTEN loss of function models in specific subsets of cells during carcinogenesis and ontogenesis.

Keywords

References

  1. Ai, J., Pascal, L.E., O'Malley, K.J., Dar, J.A., Isharwal, S., Qiao, Z., Ren, B., Rigatti, L.H., Dhir, R., Xiao, W., et al. (2014). Concomitant loss of EAF2/U19 and Pten synergistically promotes prostate carcinogenesis in the mouse model. Oncogene 33, 2286-2294. https://doi.org/10.1038/onc.2013.190
  2. Backman, S.A., Stambolic, V., Suzuki, A., Haight, J., Elia, A., Pretorius, J., Tsao, M.S., Shannon, P., Bolon, B., Ivy, G.O., et al. (2001). Deletion of Pten in mouse brain causes seizures, ataxia and defects in soma size resembling Lhermitte-Duclos disease. Nat. Genet. 29, 396-403. https://doi.org/10.1038/ng782
  3. Bernards, R. (2014). Finding effective cancer therapies through loss of function genetic screens. Curr. Opin. Genet. Dev. 24, 23-29. https://doi.org/10.1016/j.gde.2013.11.007
  4. Di Cristofano, A., Pesce, B., Cordon-Cardo, C., and Pandolfi, P.P. (1998). Pten is essential for embryonic development and tumour suppression. Nat. Genet. 19, 348-355. https://doi.org/10.1038/1235
  5. Dickins, R.A., McJunkin, K., Hernando, E., Premsrirut, P.K., Krizhanovsky, V., Burgess, D.J., Kim, S.Y., Cordon-Cardo, C., Zender, L., Hannon, G.J., et al. (2007). Tissue-specific and reversible RNA interference in transgenic mice. Nat. Genet. 39, 914-921. https://doi.org/10.1038/ng2045
  6. Fellmann, C., and Lowe, S.W. (2014). Stable RNA interference rules for silencing. Nat. Cell Biol. 16, 10-18. https://doi.org/10.1038/ncb2895
  7. Fritsch, L., Martinez, L.A., Sekhri, R., Naguibneva, I., Gerard, M., Vandromme, M., Schaeffer, L., and Harel-Bellan, A. (2004). Conditional gene knock-down by CRE-dependent short interfering RNAs. EMBO Rep. 5, 178-182. https://doi.org/10.1038/sj.embor.7400064
  8. Groszer, M., Erickson, R., Scripture-Adams, D.D., Lesche, R., Trumpp, A., Zack, J.A., Kornblum, H.I., Liu, X., and Wu, H. (2001). Negative regulation of neural stem/progenitor cell proliferation by the Pten tumor suppressor gene in vivo. Science 294, 2186-2189. https://doi.org/10.1126/science.1065518
  9. Gupta, S., Schoer, R.A., Egan, J.E., Hannon, G.J., and Mittal, V. (2004). Inducible, reversible, and stable RNA interference in mammalian cells. Proc. Natl. Acad. Sci. USA 101, 1927-1932. https://doi.org/10.1073/pnas.0306111101
  10. Hitz, C., Wurst, W., and Kuhn, R. (2007). Conditional brain-specific knockdown of MAPK using Cre/loxP regulated RNA interference. Nucleic Acids Res. 35, e90. https://doi.org/10.1093/nar/gkm475
  11. Hopkins, B.D., Hodakoski, C., Barrows, D., Mense, S.M., and Parsons, R.E. (2014). PTEN function: the long and the short of it. Trends Biochem. Sci. 39, 183-190. https://doi.org/10.1016/j.tibs.2014.02.006
  12. Im, E., Jung, J., Pothoulakis, C., and Rhee, S.H. (2014). Disruption of Pten speeds onset and increases severity of spontaneous colitis in Il10(-/-) mice. Gastroenterology 147, 667-679 e610. https://doi.org/10.1053/j.gastro.2014.05.034
  13. Ittner, L.M., and Gotz, J. (2007). Pronuclear injection for the production of transgenic mice. Nat. Protoc. 2, 1206-1215. https://doi.org/10.1038/nprot.2007.145
  14. Kasim, V., Miyagishi, M., and Taira, K. (2004). Control of siRNA expression using the Cre-loxP recombination system. Nucleic Acids Res. 32, e66. https://doi.org/10.1093/nar/gnh061
  15. Kleinhammer, A., Deussing, J., Wurst, W., and Kuhn, R. (2011). Conditional RNAi in mice. Methods 53, 142-150. https://doi.org/10.1016/j.ymeth.2010.08.003
  16. Knobbe, C.B., Lapin, V., Suzuki, A., and Mak, T.W. (2008). The roles of PTEN in development, physiology and tumorigenesis in mouse models: a tissue-by-tissue survey. Oncogene 27, 5398-5415. https://doi.org/10.1038/onc.2008.238
  17. Ko, J.K., Choi, K.H., Zhao, X., Komazaki, S., Pan, Z., Weisleder, N., and Ma, J. (2011). A versatile single-plasmid system for tissue-specific and inducible control of gene expression in transgenic mice. FASEB J. 25, 2638-2649. https://doi.org/10.1096/fj.11-181560
  18. Kunath, T. (2008). Transgenic RNA interference to investigate gene function in the mouse. Methods Mol. Biol. 461, 165-186. https://doi.org/10.1007/978-1-60327-483-8_11
  19. Kwon, C.H., Zhu, X., Zhang, J., Knoop, L.L., Tharp, R., Smeyne, R.J., Eberhart, C.G., Burger, P.C., and Baker, S.J. (2001). Pten regulates neuronal soma size: a mouse model of Lhermitte-Duclos disease. Nat. Genet. 29, 404-411. https://doi.org/10.1038/ng781
  20. Li, L., Liu, F., and Ross, A.H. (2003). PTEN regulation of neural development and CNS stem cells. J. Cell. Biochem. 88, 24-28. https://doi.org/10.1002/jcb.10312
  21. Matthess, Y., Kappel, S., Spankuch, B., Zimmer, B., Kaufmann, M., and Strebhardt, K. (2005). Conditional inhibition of cancer cell proliferation by tetracycline-responsive, H1 promoter-driven silencing of PLK1. Oncogene 24, 2973-2980. https://doi.org/10.1038/sj.onc.1208472
  22. Miething, C., Scuoppo, C., Bosbach, B., Appelmann, I., Nakitandwe, J., Ma, J., Wu, G., Lintault, L., Auer, M., Premsrirut, P.K., et al. (2014). PTEN action in leukaemia dictated by the tissue microenvironment. Nature 510, 402-406. https://doi.org/10.1038/nature13239
  23. Orloff, M.S., and Eng, C. (2008). Genetic and phenotypic heterogeneity in the PTEN hamartoma tumour syndrome. Oncogene 27, 5387-5397. https://doi.org/10.1038/onc.2008.237
  24. Premsrirut, P.K., Dow, L.E., Kim, S.Y., Camiolo, M., Malone, C.D., Miething, C., Scuoppo, C., Zuber, J., Dickins, R.A., Kogan, S.C., et al. (2011). A rapid and scalable system for studying gene function in mice using conditional RNA interference. Cell 145, 145-158. https://doi.org/10.1016/j.cell.2011.03.012
  25. Song, M.S., Salmena, L., and Pandolfi, P.P. (2012). The functions and regulation of the PTEN tumour suppressor. Nature reviews. Mol. Cell Biol. 13, 283-296. https://doi.org/10.1038/nrm3330
  26. Tiscornia, G., Tergaonkar, V., Galimi, F., and Verma, I.M. (2004). CRE recombinase-inducible RNA interference mediated by lentiviral vectors. Proc. Natl. Acad. Sci. USA 101, 7347-7351. https://doi.org/10.1073/pnas.0402107101
  27. Wilson, R.C., and Doudna, J.A. (2013). Molecular mechanisms of RNA interference. Annu. Rev. Biophys. 42, 217-239. https://doi.org/10.1146/annurev-biophys-083012-130404
  28. Wiznerowicz, M., Szulc, J., and Trono, D. (2006). Tuning silence: conditional systems for RNA interference. Nat. Methods 3, 682-688. https://doi.org/10.1038/nmeth914

Cited by

  1. Chemokine-Like Receptor 1 Regulates the Proliferation and Migration of Vascular Smooth Muscle Cells vol.22, 2016, https://doi.org/10.12659/MSM.897832