टेट्रासाइक्लिन-inducible (Tet पर) shRNA या सीडीएनए अभिव्यक्ति के साथ स्थिर मानव कोशिका लाइनों का सृजन
Summary
Inducible और प्रतिवर्ती सीडीएनए या हित के जीन की overexpression shRNA की मध्यस्थता दस्तक नीचे के साथ मानव कोशिका लाइनों उत्पन्न करने के लिए एक तेजी से और सरल तरीका है. इस विधि मज़बूती और अत्यधिक reproducibly में हेरफेर सेल लाइनों है कि क्षणिक अभिकर्मक तरीकों या पारंपरिक / पछाड़ना पीटा रणनीतियों द्वारा बदल मुश्किल हैं शोधकर्ताओं के लिए सक्षम बनाता है.
Abstract
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.
Protocol
Representative Results
Discussion
हम मानते हैं कि Tet पर सिस्टम बहुत सेल प्रणाली है कि हेरफेर करने के लिए और / या जब चालाकी से जीन सेल अस्तित्व के लिए आवश्यक है के लिए मुश्किल हैं में विशेष रूप से जीन समारोह का विश्लेषण, सुविधा. इसके अलावा, Tet ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
हम उपयोगी विचार – विमर्श के लिए हमारी प्रयोगशाला के सभी सदस्यों को धन्यवाद. हम पांडुलिपि के महत्वपूर्ण पढ़ने के लिए Joanna Lisztwan और क्रिस्टीना Gewinner धन्यवाद. इस काम बीबीएसआरसी अनुदान BB/I021248/1 द्वारा समर्थित किया गया था और वेलकम ट्रस्ट अनुदान 090090/Z/09/ZAH UCL कैंसर संस्थान में एक वेलकम ट्रस्ट रिसर्च कैरियर विकास साथी है.
Materials
| 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 |
References
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