Can the Clontech Tet-On® system or Tet-Off® system components be used with your T-REx™ tetracycline-regulated mammalian expression system?

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Answer

No. The two systems are not compatible since they utilize different strategies for promoter regulation. The T-REx™ system is designed such that native E. coli tet-repressor protein molecules bind to specific tet-operator sequences (2X TO) just downstream of the TATA box in the full length CMV promoter in the expression vector. This binding keeps the promoter silent simply by preventing the normal transcription machinery from productive assembly at the TATA box. Incidentally, it is this full length CMV promoter region that permits higher induced expression levels relative to other systems.

The recombinant ’repressor’ proteins utilized in Clontech’s system are actually recombinant fusion proteins which also contain a potent transcriptional transactivator. The Clontech system places operator sequences 5’ to the TATA box and relies upon the VP16 transactivator to promote transcription. These repressor-transactivator fusion constructs would have unpredictable and unreliable effects at the CMV promoter in our expression constructs. Additionally, the tet-repressor protein produced from the pCDNA™6/TR construct in the T-REx™ system has no transactivation domain and so would exert little regulatory effect at the minimal promoter region (non-full length CMV) found in the Clontech response plasmids.

Answer Id: E3947

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c5ed918b621c748ded60e4ba6c8c300d_FAQ

I am working with a mouse cell line and would like to express my gene at high levels using one of your vectors with the CMV promoter. Do you foresee any problems with this approach?

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Answer

The CMV promoter is known to be downregulated over time in mouse cell lines. Hence, we recommend using one of our non-CMV vectors, such as those with the EF1alpha or UbC promoter, for long-term expression in mouse cell lines.

Answer Id: E9152

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aa6000854a733631ad122a7c944854d0_FAQ

Which competent E. coli do you recommend using for propagation of my Gateway®-adapted mammalian Destination vector?

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Answer

We recommend using One Shot® ccdB Survival™ 2 T1R Competent Cells, Cat. No. A10460. This strain is resistant to the toxic effects of the ccdB gene. Note: Do not use general E. coli cloning strains, including TOP10 or DH5alpha™, for propagation and maintenance, as these strains are sensitive to ccdB effects.

Answer Id: E9153

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af510a4ec03aced844ef5cd3277b4b3d_FAQ

Do you offer a GFP-expressing mammalian expression vector that I can use as a control to monitor my transfection and expression?

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Answer

We offer pJTI™ R4 Exp CMV EmGFP pA Vector, Cat. No. A14146, which you can use to monitor your transfection and expression.

Answer Id: E9154

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2c85f53110326245a2efcdaf9c503be0_FAQ

I am interested in a mammalian expression system where I can have regulated expression of my gene of interest. I see that you offer multiple systems for this purpose. Can you describe the main features of each system?

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Answer

We offer three unique mammalian expression systems for inducible/regulated expression of the gene of interest:

- T-REx™ system
- Flp-In™ T-REx™ system
- GeneSwitch™ system

Please see below to see how they compare with one another:
System -- Basal Expression Level -- Induced Expression Level -- Response time to Maximal Expression -- Transgenic Appliation
T-Rex&trade system -- Low -- Highest -- High -- Suitable
Flp-In™ T-REx™ system -- Lower -- High -- 24-48 hrs -- Suitable
GeneSwitch™ system -- Lowest -- High -- 24-48 hrs -- Suitable

Answer Id: E9159

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e627b5e3f9cf87855482a0d02333fc3b_FAQ

Can I use the anti-HisG antibody for western detection of the His tag in Gateway® pT-REx™ DEST31?

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Answer

The Gateway® pT-REx™ DEST31 vector contains an N-terminal 6xHis tag (the 6XHis in this vector is not followed by Glycine (G) or -COOH). Hence, it cannot be detected using anti-HisG or anti-His (C-terminal) antibodies. Instead, we recommend using an anti-6xHis antibody (Cat. No. 372900).

Answer Id: E9161

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e0ac326977cf8e5be01d8ee731837746_FAQ

Can I use doxycycline instead of tetracycline as an inducer in the T-REx™ system?

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Answer

Doxycycline may be used as an alternative inducing agent in the T-REx™ system. It is similar to tetracycline in its mechanism of action, and exhibits similar dose-response and induction characteristics as tetracycline in the T-REx™ system. Doxycycline has been shown to have a longer half-life than tetracycline (48 hours vs. 24 hours, respectively). We do not offer doxycycline, but it may be obtained from Sigma (Cat. No. D9891).

Answer Id: E9163

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57495abe62359b36f2a53e02935c5d1f_FAQ

What is the advantage of the Flp-In™ T-REx™ system over the T-REx™ system?

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Answer

The Flp-In™ T-REx™ system combines the targeted integration offered by the Flp-In™ system with the powerful inducible expression offered by the T-REx™ system. It allows generation of isogenic, inducible, stable cell lines and permits polyclonal selection of these cell lines. Once the Flp-In™ T-REx™ host cell line containing an integrated FRT site has been created, subsequent generation of Flp-In™ T-REx™ cell lines expressing the gene(s) of interest is rapid and efficient.

Answer Id: E9164

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f0222bddc59de99cf61e574928982630_FAQ

What is the main advantage of the GeneSwitch™ system over the T-REx™ system? And what is its main disadvantage?

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Answer

With the GeneSwitch™ system, it is possible to have the absolute lowest basal levels of expression of the gene of interest, whereas the T-REx™ system may be a little leaky due to the inevitable presence of tetracycline in FBS. The induced level of expression in the GeneSwitch™ system can be even higher than that seen with the CMV promoter. The disadvantage of the GeneSwitch™ system is that the expression does not appear to switch off very easily in culture, although it has been demonstrated to function beautifully in transgenics. The T-REx™ system, on the other hand, can be switched on and off by the addition and removal of the inducer.

Answer Id: E9166

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915bb83307fd2c5cc59afc9d88ef73f4_FAQ

Why is sequential transfection recommended over co-transfection in the T-REx™ and GeneSwitch™ systems?

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Answer

When a co-transfection is performed, there is no way of testing the double stable cell line for functional TetR or GeneSwitch™ protein, respectively. On the other hand, when sequential transfection is performed, one can functionally test the generated T-REx™ or GeneSwitch™ cell line by transiently transfecting the lacZ expression control plasmid and then picking a clone that shows the lowest basal level of expression of lacZ in the absence of the inducer, and the highest level of lacZ in the presence of the inducer. This clone can then be expanded and used to transfect the T-REx™ or GeneSwitch™ expression construct, as the case may be.

Answer Id: E9167

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4fa7d7bf6e48356fcc5814995e81e9e5_FAQ

Is it okay if my construct has an ATG that is upstream of the ATG in my gene of interest? Will it interfere with translation of my gene?

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Answer

Translation initiation will occur at the first ATG encountered by the ribosome, although in the absence of a Kozak sequence, initiation will be relatively weak. Any insert downstream would express a fusion protein if it is in frame with this initial ATG, but levels of expressed protein are predicted to be low if there is a non-Kozak consensus sequence. If the vector contains a non-Kozak consensus ATG, we recommend that you clone your gene upstream of that ATG and include a Kozak sequence for optimal expression.

Answer Id: E9180

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16c54268c5857e0549e9da663fb0c2ef_FAQ

I am using a mammalian expression vector that has the neomycin resistance gene. Can I use neomycin for stable selection in mammalian cells?

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Answer

No; neomycin is toxic to mammalian cells. We recommend using Geneticin® (a.k.a. G418 Sulfate), as it is a less toxic and very effective alternative for selection in mammalian cells.

Answer Id: E9181

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86410c89d41682a89d98002342b5c3f1_FAQ

I used a mammalian expression vector but do not get any expression of my protein. Can you help me troubleshoot?

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Answer

Here are possible causes and solutions:

- Try the control expression that is included in the kit
Possible detection problem:

- Detection of expressed protein may not be possible in a transient transfection, since the transfection efficiency may be too low for detection by methods that assess the entire transfected population. We recommend optimizing the transfection efficiency, doing stable selection, or using methods that permit examination of individual cells. You can also increase the level of expression by changing the promoter or cell type.
- Expression within the cell may be too low for the chosen detection method. We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot. Protein might be degraded or truncated: Check on a Northern. Possible time-course issue: Since the expression of a protein over time will depend upon the nature of the protein, we always recommend doing a time course for expression. A pilot time-course assay will help to determine the optimal window for expression. Possible cloning issues: Verify clones by restriction digestion and/or sequencing.

Answer Id: E9182

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78bbc5bb321b11c2f6a40a2e143aae2a_FAQ

I performed stable selection but my antibiotic-resistant clones do not express my gene of interest. What could have gone wrong?

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Answer

Here are possible causes and solutions:

Detection method may not be appropriate or sensitive enough:
- We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot.
- Insufficient number of clones screened: Screen at least 20 clones.
- Inappropriate antibiotic concentration used for stable selection: Make sure the antibiotic kill curve was performed correctly. Since the potency of a given antibiotic depends upon cell type, serum, medium, and culture technique, the dose must be determined each time a stable selection is performed. Even the stable cell lines we offer may be more or less sensitive to the dose we recommend if the medium or serum is significantly different.
- Expression of gene product (even low level) may not be compatible with growth of the cell line: Use an inducible expression system.
- Negative clones may result from preferential linearization at a vector site critical for expression of the gene of interest: Linearize the vector at a site that is not critical for expression, such as within the bacterial resistance marker.

Answer Id: E9183

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559c6028788610ad1cbc4bc184da429d_FAQ

Do you offer Gateway® vectors for expression in plants?

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Answer

We do not offer any Gateway® vectors for expression in plants.

Answer Id: E9854

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f35a2a2fda93397e37ba9cec45315b52_FAQ