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Biology

Generation of Stable Human Cell Lines with Tetracycline-inducible (Tet-on) shRNA or cDNA Expression

Published: March 5th, 2013

DOI:

10.3791/50171

1UCL Cancer Institute, 2Friedrich Miescher Institute for Biomedical Research

A rapid and simple way to generate human cell lines with inducible and reversible cDNA overexpression or shRNA-mediated knock-down of the gene of interest. This method enables researchers to reliably and highly reproducibly manipulate cell lines that are difficult to alter by transient transfection methods or conventional knockdown/knockout strategies.

A major approach in the field of mammalian cell biology is the manipulation of the expression of genes of interest in selected cell lines, with the aim to reveal one or several of the gene's function(s) using transient/stable overexpression or knockdown of the gene of interest. Unfortunately, for various cell biological investigations this approach is unsuitable when manipulations of gene expression result in cell growth/proliferation defects or unwanted cell differentiation. Therefore, researchers have adapted the Tetracycline repressor protein (TetR), taken from the E. coli tetracycline resistance operon1, to generate very efficient and tight regulatory systems to express cDNAs in mammalian cells2,3. In short, TetR has been modified to either (1) block initiation of transcription by binding to the Tet-operator (TO) in the promoter region upon addition of tetracycline (termed Tet-off system) or (2) bind to the TO in the absence of tetracycline (termed Tet-on system) (Figure 1). Given the inconvenience that the Tet-off system requires the continuous presence of tetracycline (which has a half-life of about 24 hr in tissue cell culture medium) the Tet-on system has been more extensively optimized, resulting in the development of very tight and efficient vector systems for cDNA expression as used here.

Shortly after establishment of RNA interference (RNAi) for gene knockdown in mammalian cells4, vectors expressing short-hairpin RNAs (shRNAs) were described that function very similar to siRNAs5-11. However, these shRNA-mediated knockdown approaches have the same limitation as conventional knockout strategies, since stable depletion is not feasible when gene targets are essential for cellular survival. To overcome this limitation, van de Wetering et al.12 modified the shRNA expression vector pSUPER5 by inserting a TO in the promoter region, which enabled them to generate stable cell lines with tetracycline-inducible depletion of their target genes of interest.

Here, we describe a method to efficiently generate stable human Tet-on cell lines that reliably drive either inducible overexpression or depletion of the gene of interest. Using this method, we have successfully generated Tet-on cell lines which significantly facilitated the analysis of the MST/hMOB/NDR cascade in centrosome13,14 and apoptosis signaling15,16. In this report, we describe our vectors of choice, in addition to describing the two consecutive manipulation steps that are necessary to efficiently generate human Tet-on cell lines (Figure 2). Moreover, besides outlining a protocol for the generation of human Tet-on cell lines, we will discuss critical aspects regarding the technical procedures and the characterization of Tet-on cells.

1. Cloning of pcDNA6_TetR_IRES_blast

  1. As illustrated in Figure 3, perform a partial digest of the pcDNA6/TR plasmid (V1025-20, Invitrogen) with the restriction enzymes XbaI and NcoI to remove the TetR gene and the promoter of the blasticidin resistance (BlastR) gene. Isolate the 4.6 kb vector fragment.
  2. To remove the polyadenylation sequence from the TetR gene, introduce by PCR an XhoI site immediately after the Stop codon of the TetR cDNA while conserving t.......

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An example for the initial characterization of RPE-1 cell lines stably expressing TetR is shown in Figure 4. Note that all RPE-1 clones express varying levels of TetR (compare lanes 2 and 5), while the parental cell line (which serves as negative control) does not express the exogenous TetR protein (Figure 4, lane 1). This variation in TetR expression among RPE-1 Tet-on cell clones is expected, since the expression of the TetR expressing plasmids is highly dependent on the plasmid.......

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We believe that Tet-on systems greatly facilitate the analysis of gene function, particularly in cell systems that are difficult to manipulate and/or when the manipulated gene is essential for cell survival. Furthermore, the ability to control gene expression by highly specific Tet-on systems offers the opportunity to study gene functions at different stages (for example during cell cycle progression or well-defined differentiation processes). The method presented here will enable researchers to generate the desir.......

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We thank all members of our laboratory for helpful discussions. We thank Joanna Lisztwan and Christina Gewinner for critical reading of the manuscript. This work was supported by the BBSRC grant BB/I021248/1 and the Wellcome Trust grant 090090/Z/09/Z.A.H. is a Wellcome Trust Research Career Development fellow at the UCL Cancer Institute.

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Name Company Catalog Number Comments
Name of the reagent Company Catalogue number Comments (optional)
Fetal Bovine Serum(FBS) Invitrogen 16000-044 Tested Tet-free
Blasticidin Invivogen ant-bl-1
Zeocin Invivogen ant-zn-5
G418 PAA laboratories P31-011 100 mg/ml in media
anti-TET02 MoBiTec GmbH TET02 Use at 1/1000 to 1/2000 for WB
pcDNA6/TR Invitrogen V1025-20
pT-Rex DEST30 Invitrogen 12301-016
Tetracycline Sigma 87128 2 mg/ml in ethanol
Doxycycline Sigma D9891 2 mg/ml in water
Cloning cylinders Bellco Glass Inc. 2090-00808 re-useable

  1. Postle, K., Nguyen, T. T., Bertrand, K. P. Nucleotide sequence of the repressor gene of the TN10 tetracycline resistance determinant. Nucleic Acids Res. 12, 4849-4863 (1984).
  2. Gossen, M., Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. U.S.A. 89, 5547-5551 (1992).
  3. Gossen, M., et al. Transcriptional activation by tetracyclines in mammalian cells. Science. 268, 1766-1769 (1995).
  4. Elbashir, S. M., et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 411, 494-498 (2001).
  5. Brummelkamp, T. R., Bernards, R., Agami, R. A system for stable expression of short interfering RNAs in mammalian cells. Science. 296, 550-553 (2002).
  6. McManus, M. T., Petersen, C. P., Haines, B. B., Chen, J., Sharp, P. A. Gene silencing using micro-RNA designed hairpins. RNA. 8, 842-850 (2002).
  7. Miyagishi, M., Taira, K. U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells. Nat. Biotechnol. 20, 497-500 (2002).
  8. Paddison, P. J., Caudy, A. A., Bernstein, E., Hannon, G. J., Conklin, D. S. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev. 16, 948-958 (2002).
  9. Paul, C. P., Good, P. D., Winer, I., Engelke, D. R. Effective expression of small interfering RNA in human cells. Nat. Biotechnol. 20, 505-508 (2002).
  10. Sui, G., et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 99, 5515-5520 (2002).
  11. Yu, J. Y., DeRuiter, S. L., Turner, D. L. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 99, 6047-6052 (2002).
  12. van de Wetering, M., et al. Specific inhibition of gene expression using a stably integrated, inducible small-interfering-RNA vector. EMBO Rep. 4, 609-615 (2003).
  13. Hergovich, A., et al. The MST1 and hMOB1 tumor suppressors control human centrosome duplication by regulating NDR kinase phosphorylation. Curr. Biol. 19, 1692-1702 (2009).
  14. Hergovich, A., Lamla, S., Nigg, E. A., Hemmings, B. A. Centrosome-associated NDR kinase regulates centrosome duplication. Mol. Cell. 25, 625-634 (2007).
  15. Kohler, R. S., Schmitz, D., Cornils, H., Hemmings, B. A., Hergovich, A. Differential NDR/LATS interactions with the human MOB family reveal a negative role for human MOB2 in the regulation of human NDR kinases. Mol. Cell Biol. 30, 4507-4520 (2010).
  16. Vichalkovski, A., et al. NDR kinase is activated by RASSF1A/MST1 in response to Fas receptor stimulation and promotes apoptosis. Curr. Biol. 18, 1889-1895 (2008).
  17. Pear, W. S., et al. Efficient and rapid induction of a chronic myelogenous leukemia-like myeloproliferative disease in mice receiving P210 bcr/abl-transduced bone marrow. Blood. 92, 3780-3792 (1998).

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