Name: generation of stable human cell lines with tetracycline-inducible (tet-on) shrna or cdna expression
Uploaded: 2013-03-05T13:00:00
Description: Watch this Scientific Journal Video about Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression at JoVE.com

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.

1. Cloning of pcDNA6_TetR_IRES_blast
<ol>
	<li>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.</li>
	<li>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...

<ol>
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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...

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

generation of stable human cell lines with tetracycline-inducible (tet-on) shrna or cdna expression

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.

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...

Watch this Scientific Journal Video about Generation of Stable Human Cell Lines with Tetracycline-inducible Tet-on shRNA or cDNA Expression at JoVE.com

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

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.

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

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 ...

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