pcDNA™4/TO Mammalian Expression Vector

Citations & References (5)

Invitrogen™

pcDNA™4/TO Mammalian Expression Vector

A Tetracycline-Regulated Expression System without Viral TransactivatorsThe T-REx™ System yields higher levels of induced expression than any other regulated mammalianRead more

Catalog Number	Quantity
V102020 also known as V1020-20	20 μg

Catalog number V102020

also known as V1020-20

Price (CLP)

Quantity:

20 μg

A Tetracycline-Regulated Expression System without Viral Transactivators
The T-REx™ System yields higher levels of induced expression than any other regulated mammalian expression system. It utilizes the complete CMV promoter and adds control elements from the bacterial tetracycline resistance operon to effectively repress and derepress transcription from one of the strongest mammalian promoter sequences known (1,2).

Specific Activation
The T-REx™ System uses a repressor mechanism that blocks transcription from the powerful CMV promoter in the absence of tetracycline. Because the T-REx™ System elements do not use viral transactivators, you can achieve high-level expression from the complete CMV promoter without secondary, non-specific activation of host genes.

The T-REx™ Mechanism
The T-REx™ transcriptional control elements are illustrated in Figure 1. Two tetracycline operator sequences (TetO₂) have been inserted between the TATA box of the CMV promoter and the transcriptional start site. The TetO₂ sequence itself has no effect on expression. When the tetracycline repressor protein (TR) is present, it effectively binds the TetO₂ sites and blocks transcription initiation. Tetracycline added to the culture medium binds to, and changes the conformation of, the TR protein. This change causes the TR protein to release the TetO₂ sites, derepressing transcription from the CMV promoter. The result is high-level expression of the gene of interest (Figure 2). Expression levels can be modulated based on the tetracycline concentration and can be induced to levels that are achieved with constitutive CMV expression vectors.
T-REx™ is a powerful inducible mammalian expression system that allows you to regulate expression from the complete human cytomegalovirus (CMV) enhancer-promoter. T-REx™inducible expression vectors offer the following features:

• Complete CMV enhancer-promoter sequence containing two copies of the tetracycline operator TetO₂ sequence for high-level regulated expression
• Zeocin™ or hygromycin resistance gene for effective selection of stable mammalian cell lines
• Large multiple cloning site to simplify cloning

In addition, pcDNA™4/TO/myc-His offers a c-myc epitope for rapid detection of the recombinant protein with an Anti-myc Antibody and a polyhistidine (6xHis) sequence for simple purification of the recombinant protein with nickel-chelating resin and detection with Anti- His(C-term) Antibody.

The regulatory vector, pcDNA™6/TR, is provided for high-level expression of the tetracycline repressor (TR) protein. This vector expresses the Blasticidin resistance gene for rapid selection of mammalian cell lines that stably express the TR protein.

For Research Use Only. Not for use in diagnostic procedures.

Specifications

Constitutive or Inducible SystemInducible

Delivery TypeTransfection

For Use With (Application)Regulated Expression

Inducing AgentTetracycline

Product TypeMammalian Expression Vector

Quantity20 μg

Selection Agent (Eukaryotic)Zeocin™

VectorpcDNA

Cloning MethodRestriction Enzyme/MCS

Product LineT-REx, pcDNA

PromoterCMV/TO

Protein TagUntagged

Unit Size20 µg

Frequently asked questions (FAQs)

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

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.

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

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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

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.

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?

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.

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

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

View all pcDNA™4/TO Mammalian Expression Vector FAQs

Citations & References (5)

Citations & References

Abstract

Retention of mutant low density lipoprotein receptor in ER leads to ER stress.

Authors:Sørensen S, Ranheim T, Bakken KS, Leren TP, Kulseth MA,

Journal:J Biol Chem

PubMed ID:16257961

Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the gene encoding the low density lipoprotein receptor (LDLR). More than 50% of these mutations lead to receptor proteins that are completely or partly retained in the endoplasmic reticulum (ER). The mechanisms involved in the intracellular processing and ... More

BID-D59A is a potent inducer of apoptosis in primary embryonic fibroblasts.

Authors:Sarig R, Zaltsman Y, Marcellus RC, Flavell R, Mak TW, Gross A,

Journal:J Biol Chem

PubMed ID:12519725

The proapoptotic activity of BID seems to solely depend upon its cleavage to truncated tBID. Here we demonstrate that expression of a caspase-8 non-cleavable (nc) BID-D59A mutant or expression of wild type (wt) BID induces apoptosis in Bid -/-, caspase-8 -/-, and wt primary MEFs. Western blot analysis indicated that ... More

Cell Cycle Regulation and p53 Activation by Protein Phosphatase 2Calpha.

Authors:Ofek P, Ben-Meir D, Kariv-Inbal Z, Oren M, Lavi S,

Journal:J Biol Chem

PubMed ID:12514180

Protein phosphatase 2C (PP2C) dephosphorylates a broad range of substrates, regulating stress response and growth-related pathways in both prokaryotes and eukaryotes. We now demonstrate that PP2Calpha, a major mammalian isoform, inhibits cell growth and activates the p53 pathway. In 293 cell clones, in which PP2Calpha expression is regulated by a ... More

The gamma -secretase-cleaved C-terminal fragment of amyloid precursor protein mediates signaling to the nucleus.

Authors: Gao Y; Pimplikar S W;

Journal:Proc Natl Acad Sci U S A

PubMed ID:11742091

Sequential processing of the amyloid precursor protein (APP) by beta- and gamma-secretases generates the Abeta peptide, a major constituent of the senile plaques observed in Alzheimer's disease. The cleavage by gamma-secretase also results in the cytoplasmic release of a 59- or 57-residue-long C-terminal fragment (Cgamma). This processing resembles regulated intramembrane ... More

ATR is not required for p53 activation but synergizes with p53 in the replication checkpoint.

Authors: Nghiem Paul; Park Peter K; Kim Ys Yong-son; Desai Bimal N; Schreiber Stuart L;

Journal:J Biol Chem

PubMed ID:11711532

ATR (ataxia telangiectasia and Rad-3-related) is a protein kinase required for survival after DNA damage. A critical role for ATR has been hypothesized to be the regulation of p53 and other cell cycle checkpoints. ATR has been shown to phosphorylate p53 at Ser(15), and this damage-induced phosphorylation is diminished by ... More