Invitrogen™ GeneSwitch™ Mammalian Expression Kit, complete kit

Catalog No.	Quantity
K106001	1 kit

Catalog No. K106001 Supplier Invitrogen™ Supplier No. K106001

Description

Includes

20μg of each of the following vectors: pGene/V5-His (A, B, and C), the expression control vector pGene/V5-His/lacZ, and the pSwitch vector. The kit also includes forward and reverse sequencing primers and 100μg of the inducing agent mifepristone. The GeneSwitch Complete Kit includes the Core Kit with 1g hygromycin and 1g Zeocin.

Offers exceptionally low uninduced and high induced expression levels in mammalian cells

• Expression vector pGene/V5-His provides minimal promoter, GAL4-E1b, consisting of binding sites for yeast Gal4 DNA binding protein followed by TATA sequence from Adenovirus E1b promoter
• Without additional factors, GAL4-E1b promoter is transcriptionally silent
• To activate transcription from GAL4-E1b promoter, GeneSwitch regulatory protein is expressed from minimal TK promoter on pSwitch vector
• GeneSwitch protein has three functional domains: Gal4 DNA Binding Domain (Gal4-DBD) to bind regulatory protein to GAL4-E1b promoter; Truncated human Progesterone Receptor Ligand Binding Domain (hPR-LBD) that undergoes a conformational change when it binds the progesterone antagonist, mifepristone; Transcription activation domain from NFØB transcription factor p65 (p65AD) to activate transcription from silent GAL4-E1b promoter
• In absence of mifepristone, conformation of hPR-LBD region prevents GeneSwitch regulatory protein from activating transcription from GAL4-E1b promoter
• When mifepristone is added and binds hPR-LBD region, GeneSwitch regulatory protein assumes conformation that permits to stimulate transcription from GAL4-E1b promoter
• Induction of GeneSwitch System leads to activation of gene of interest on vector pGene/V5-His
• In addition, four Gal4 binding sites upstream of minimal HSV TK promoter on pSwitch can bind pSwitch regulatory protein
• Therefore, adding mifepristone up-regulates production of regulatory protein
• Increased levels of GeneSwitchregulatory protein result in induction of gene of interest from pGene/V5-His to levels that can approach those of viral promoters
• Provides expression vector pGene/V5-His with minimal, synthetic GAL4-E1b promoter that is transcriptionally silent until activated

• In addition, GeneSwitch protein upregulates its own expression, leading to amplified expression of gene of interest from pGene/V5-His
• C-terminal V5 epitope tag for detection with Anti-V5 Antibodies
• C-terminal polyhistidine (6xHis) tag for rapid purification with nickel-chelating resin and detection with Anti-His(C-term) Antibodies
• Zeocin resistance gene for effective selection in both mammalian cells and E. coli

Mammalian Expression, Protein Expression, Proteins, Expression, Isolation and Analysis, Regulated Expression

Specifications

• Constitutive or Inducible System: Inducible
• Delivery Type: Transfection
• Inducing Agent: Mifepristone
• Promoter: E1b (GAL4-E1b)
• Product Type: Mammalian Expression Kit
• Selection Agent (Eukaryotic): Zeocin™
• Content And Storage: The GeneSwitch™ System Core Kit includes 20 μg of each of the following vectors: pGene/V5-His (A, B, and C), the expression control vector pGene/V5-His/lacZ, and the pSwitch vector. The kit also includes forward and reverse sequencing primers and 100 μg of the inducing agent mifepristone. The GeneSwitch™ Complete Kit includes the Core Kit with 1 g hygromycin and 1 g Zeocin™ at considerable savings. Hygromycin should be stored at +4°C and Zeocin™ at -20°C. Zeocin™ should be protected from exposure to light. All reagents are stable for 6 months when properly stored.
• Protein Tag: His Tag (6x), V5 Epitope Tag
• Cloning Method: Restriction Enzyme/MCS
• Quantity: 1 kit
• Vector: pGene
• Product Line: GeneSwitch
• For Use With (Application): Regulated Expression

Frequently Asked Questions (FAQs)

Do you offer any GeneSwitch cell lines?

Sorry, all GeneSwitch cell lines have been discontinued.

What are the characteristic features of the GeneSwitch protein?

The GeneSwitch protein contains functional domains from different transcription factors, allowing it to function as a ligand-dependent transcription factor to activate expression of both the gene of interest and its own gene. The GeneSwitch protein exhibits the following characteristics:

- Since the GAL4 DBD is derived from a yeast protein, the GeneSwitch protein has no effect on endogenous genes and can only activate transcription of genes whose expression is controlled by a GAL4 UAS (i.e., the gene of interest and the regulatory fusion gene).
- The GAL4 DBD binds to an individual 17-nucleotide GAL4-binding site as a homodimer. The pGene/V5-His and pSwitch plasmids contain 6 and 4 copies of the GAL4 binding site, respectively, although it is not known if all of the GAL4-binding sites are occupied at any given time.
- The truncated hPR-LBD contains a 19 amino acid deletion from its C-terminal end that abolishes its ability to bind to progesterone, other endogenous steroid hormones, or other progesterone agonists, but still enables it to bind with high affinity to mifepristone.
- The p65 AD is a strong transcriptional activator but is derived from a human protein, to minimize possible toxic or pleiotropic effects associated with viral transactivation domains.

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

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.

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

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.

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?

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 system -- Low -- Highest -- High -- Suitable
Flp-In T-REx system -- Lower -- High -- 24-48 hrs -- Suitable
GeneSwitch system -- Lowest -- High -- 24-48 hrs -- Suitable

Do I need to include a ribosomal binding site (RBS/Shine Dalgarno sequence) or Kozak sequence when I clone my gene of interest?

ATG is often sufficient for efficient translation initiation although it depends upon the gene of interest. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a Shine Dalgarno sequence/RBS or consensus Kozak sequence (ACCAUGG), as the case may be. In general, all expression vectors that have an N-terminal fusion will already have a RBS or initiation site for translation.

Can you tell me the difference between a Shine-Dalgarno sequence and a Kozak sequence?

Prokaryotic mRNAs contain a Shine-Dalgarno sequence, also known as a ribosome binding site (RBS), which is composed of the polypurine sequence AGGAGG located just 5’ of the AUG initiation codon. This sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA. Similarly, eukaryotic (and specifically mammalian) mRNA also contains sequence information important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:

- Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
- Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
- Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
- Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
- Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.

Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:

- Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
- Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.

I sequenced one of your vectors after PCR amplification and observed a difference from what is provided online (or in the manual). Should I be concerned?

Our vectors have not been completely sequenced. Your sequence data may differ when compared to what is provided. Known mutations that do not affect the function of the vector are annotated in public databases.

Are your vectors routinely sequenced?

No, our vectors are not routinely sequenced. Quality control and release criteria utilize other methods.

How was the reference sequence for your vectors created?

Sequences provided for our vectors have been compiled from information in sequence databases, published sequences, and other sources.

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