Invitrogen™ pSCREEN-iT™/lacZ-DEST Gateway™ Vector Kit

Catalog No.	Quantity
V47020	20 Reactions

Catalog No. V47020 Supplier Invitrogen™ Supplier No. V47020

Description

For RNAi target screening

Use the pSCREEN-iT™/lacZ-DEST Gateway™ Vector Kit to assess the potency of any RNAi knockdown reagent using a fast, sensitive, and cost-effective β-gal reporter assay or identify the best knockdown reagent for RNAi experiments without knowing protein function, western blotting, or qRT-PCR.
How it Works: The BLOCK-iT RNAi Target Screening system method uses a β-galactosidase activity assay to measure the knockdown ability of an RNAi reagent (Stealth RNAi™ siRNA, siRNA, dicer pools, or shRNA-containing plasmid). To identify the most effective knockdown reagent for your target, simply clone your target gene into the pSCREEN-iT /lacZ-DEST Gateway vector. Co-transfect the resulting construct with the knockdown reagent being tested. Expressed genes will be fused to β-galactosidase, enabling you to use β-galactosidase levels to measure the amount of RNAi-induced degradation of the target gene. The more effective any specific knockdown reagent is at mediating RNAi, the more degradation of the fusion mRNA will occur, lowering β-gal activity.
The BLOCK-iT RNAi Target Screening System includes the pSCREEN-iT /lacZ-DEST Gateway vector for generating a construct containing a lacZ/gene of interest fusion. This vector features:

• attR sites immediately following the lacZ ORF for fast and efficient recombination with any attL-flanked Gateway entry vector to generate the lacZ/target gene fusions
• The CMV promoter for strong expression of the lacZ/target gene fusion, leading to increased assay sensitivity and better discrimination between knockdown reagents of varied effectiveness
• Positional cloning to fuse a gene or fragment to the 3' end of the lacZ gene so that β-galactosidase will be expressed even if the target fragment being tested contains stop codons

In addition, the pSCREEN-iT /lacZ-DEST vector is fully compatible with the Ultimate™ ORF Clone Collection. This makes a wide selection of potential target genes readily available for generating lacZ fusions.

RNAi, RNAi, Epigenetics and Non-Coding RNA Research, Vector-Based RNAi

Specifications

• Format: Kit
• Promoter: CMV
• Product Type: β-Galactosidase Reporter Gene Assay System
• Reporter Gene: Beta-Gal (lacZ)
• Content And Storage: The pSCREEN-iT™ /lacZ-DEST Gateway™ Vector Kit contains 6 μg of vector and 10 μg of pSCREEN-iT™/lacZ-GW/CDK2 control vector, the positive control Stealth™RNAi lacZ, a negative control Scrambled Stealth RNAi™ siRNA, and 1X annealing/dilution buffer. Store at -20°C. The BLOCK-iT™ RNAi Target Screening Kit contains the pSCREEN-iT™/lacZ-DEST Gateway™ Vector Kit, 250 μl of Lipofectamine™ 2000 Reagent, and the FluoReporter™ lacZ/Galactosidase Quantification Kit. Store the Lipofectamine™ 2000 Reagent at +4°C. Store the FluoReporter™ Kit at -20°C protected from light. The BLOCK-iT™ RNAi Target Screening System contains the BLOCK-iT™ RNAi Target Screening Kit, LR Clonase™ enzyme mix, and pCR ¤ 8/GW/TOPO ¤ TA Cloning Kit. Store the LR Clonase™ enzyme mix at -80°C. The pCR™ 8/GW/TOPO™ TA Cloning Kit includes the ready-to-use pCR™ 8/GW/TOPO™ vector, dNTPs, salt solution, water, forward and reverse sequencing primers, control template and PCR primers, One Shot™ TOP10 Chemically Competent E. coli, S.O.C. medium, and a pUC19 control plasmid. Store the Competent Cells at -80°C. Store all other components at -20°C. All components are guaranteed stable for 6 months when properly stored.
• Sample Type: Synthetic siRNA, RNAi Plasmids (shRNA, miR)
• Quantity: 20 Reactions
• Product Line: BLOCK-iT, Gateway
• For Use With (Application): RNAi Target Screening

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.

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.

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

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.

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?

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.

Do I need to include a consensus Kozak sequence when I clone my gene of interest into one of your mammalian expression vectors?

The consensus Kozak sequence is A/G NNATGG, where the ATG indicates the initiation codon. Point mutations in the nucleotides surrounding the ATG have been shown to modulate translation efficiency. Although we make a general recommendation to include a Kozak consensus sequence, the necessity depends on the gene of interest and often, the ATG alone may be sufficient for efficient translation initiation. 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 consensus Kozak. In general, all expression vectors that have an N-terminal fusion will already have an initiation site for translation.

What is the consensus Kozak sequence and what is the function of the Kozak sequence?

Eukaryotic (and specifically mammalian) mRNA contains sequence information that is 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.

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