How should I store lentivirus, adenovirus and viral vectors?

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Viral vectors:
Store lentiviral and adenoviral expression vectors at -20 degrees C. Due to their relatively large sizes, we do not recommend storing these vectors at -80 degrees C, as the vector solutions will completely freeze and too many freeze thaws from -80 degrees C will affect the cloning efficiency. At -20 degrees C, the vectors will be stable but will not freeze completely.

Virus:
Both adenovirus and lentivirus should be aliquoted immediately after production and stored at -80 degrees C.

Lentivirus is more sensitive to storage temperature and to freeze/thaw than adenovirus and should be handled with care. Adenovirus can typically be frozen/thawed up to 3 times without loss of titer, while lentivirus can lose up to 5% or more activity with each freeze/thaw. It is recommended to aliquot your virus into small working volumes immediately after production, freeze at -80 degrees C, and then thaw just one aliquot for titering. This way, every time you thaw a new aliquot it should be the same titer as your first tube.

Adenovirus can be kept overnight at 4 degrees C if necessary, but it is best to avoid this. Viruses will be most stable at -80 degrees C.

When stored properly, viral stocks should maintain consistent titer and be suitable for use for up to one year. After long-term storage, we recommend re-titering your viral stocks before use.

Answer Id: E4100

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Do you recommend a specific FBS for culture of the 293FT or 293A cells used in the ViraPower™ kits? What plastic plates do you recommend?

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We use mycoplasma-tested Gibco™ FBS (Cat. No. 16000-044) without any modifications. We have observed that when 293FT cells are cultured in the presence of this FBS following the instructions in the manual, virus production is better than that obtained with many other serum sources.

We use the following plasticware for 293A and 293FT cells:

T175--Fisher Cat. No. 10-126-13; this is a Falcon flask with 0.2 μm vented plug seal cap.

T75--Fisher Cat. No. 07-200-68; this is a Costar flask with 0.2 μm vented seal cap.

100 mm plate--Fisher Cat. No. 08-772E; this is a Falcon tissue culture-treated polystyrene plate

We get excellent adherence on these plates under routine cell culture/maintenance conditions (expect cell lysis in 293A cells when making adenovirus).

Answer Id: E4101

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

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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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I’m having difficulty sequencing the ds oligo insert in my shRNA construct. What is causing this, and do you have any suggestions on how to improve my sequencing results?

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Difficulties sequencing could occur because the hairpin sequence is an inverted repeat that can form secondary structure during sequencing, resulting in a drop in the sequencing signal when entering the hairpin. If you encounter difficulties while sequencing, please try the following:

- Use high-quality, purified plasmid DNA for sequencing. We recommend preparing DNA using the Invitrogen™ PureLink™ HQ Mini Plasmid Purification Kit (Cat. No. K2100-01) or S.N.A.P.™ Plasmid DNA MidiPrep Kit (Cat. No. K1910-01).
- Add DMSO to the sequencing reaction to a final concentration of 5%.
- Increase the amount of template used in the reaction (up to twice the normal concentration).
- Standard sequencing kits typically use dITP in place of dGTP to reduce G:C compression. Other kits containing dGTP are available for sequencing G-rich and GT-rich templates. If you are using a standard commercial sequencing kit containing dITP, obtain a sequencing kit containing dGTP (e.g., dGTP BigDye™ Terminator v3.0 Ready Reaction Cycle Sequencing Kit, Cat. No. 4390229) and use a 7:1 molar ratio of dITP:dGTP in your sequencing reaction.

Answer Id: E10019

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Will I get the same transduction efficiency with both lentivirus and adenovirus in the same cell line?

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This depends entirely on the target cell. Adenovirus requires the coxsackie-adenovirus receptor (CAR) and an integrin for efficient transduction. Lentivirus (with VSV-G) binds to a lipid in the plasma membrane (present on all cell types). With two totally different mechanisms of entry into the cell, there will always be differences in transduction efficiencies. However, the efficiency of transduction for both viral systems is easily modulated by the multiplicity of infection (MOI) used.

Answer Id: E4102

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Can I perform the single-step protocol for the BP/LR Clonase™ reaction using BP Clonase™ enzyme and LR Clonase™ enzyme instead of BP Clonase™ II enzyme and LR Clonase™ II enzyme?

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Answer

In the single-step protocol for the BP/LR Clonase™ reaction, we would not recommend substituting the BP Clonase™ II/LR Clonase™ II enzymes with BP Clonase™ /LR Clonase™ enzymes as this would result in very low recombination efficiency.

Answer Id: E9858

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I’m seeing cytotoxic effects after transfection of my shRNA/miRNA construct. What is causing this?

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Answer

You can try to scale back the amount of transfection reagent used, or use a different reagent for the transfection. Additionally, ensure that the plasmid used is pure and properly prepared for transfection.

Answer Id: E10020

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I’m seeing a low level of gene knockdown or no gene knockdown. What can you suggest I try?

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Low expression levels can be due to several factors. Please see the suggestions below:

- Low transfection efficiency: ensure that antibiotics are not added to the media during transfection, and that cells are at the proper cell confluency; optimize transfection conditions by varying the amount of transfection reagent used.
- Try a time course assay to determine the point at which the highest degree of gene knockdown occurs.
- Mutations are present in your construct: analyze the transformants by sequencing the ds oligo insert to verify its sequence.
- Target region is not optimal: select a different target region.
- Ensure siRNA is designed according to guidelines listed in the respective manual.

Answer Id: E10021

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What are the safety features built into the BLOCK-iT™ Adenoviral RNAi Expression System?

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The BLOCK-iT™ Adenoviral RNAi Expression System includes the following features designed to enhance its biosafety:

- The entire E1 region is deleted in the pAd/BLOCK-iT™-DEST expression vector. Expression of the E1 proteins is required for the expression of the other viral genes (e.g., late genes), and thus viral replication only occurs in cells that express E1 (Graham et al., 1977; Kozarsky and Wilson, 1993; Krougliak and Graham, 1995). This is where the Gateway™ Destination cassette is now located. The E3 region has also been deleted.
- Adenovirus produced from the pAd/BLOCK-iT™-DEST expression vector is replication-incompetent in any mammalian cells that do not express the E1a and E1b proteins (Graham et al., 1977; Kozarsky and Wilson, 1993; Krougliak and Graham, 1995).
- Adenovirus does not integrate into the host genome upon transduction. Because the virus is replication-incompetent, the presence of the viral genome is transient and will eventually be diluted out as cell division occurs.
- Despite the presence of the safety features discussed above, the adenovirus produced with this System can still pose some biohazardous risk since it can transduce primary human cells. For this reason, we highly recommend that you treat adenoviral stocks generated using this System as Biosafety Level 2 (BL-2) organisms and strictly follow all published guidelines for BL-2. Furthermore, exercise extra caution when creating adenovirus that express shRNA targeting human genes involved in controlling cell division (e.g., tumor suppressor genes) or when producing large-scale preparations of virus (See manual, pg. 11).
- For more information about the BL-2 guidelines and adenovirus handling, refer to the document, “Biosafety in Microbiological and Biomedical Laboratories”, 4th Edition, published by the Centers for Disease Control (CDC) (http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm)

Answer Id: E10013

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I’m trying to anneal my oligos to create a ds oligo for ligation into one of your shRNA or miRNA RNAi vectors. When I run my ligated ds oligo on an agarose gel, I do not see any bands representing the ds oligo. What could be happening?

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Answer

- Verify that the sequence of the bottom-strand oligo is complementary to the sequence of the top-strand oligo.
- For the shRNA vectors, make sure that you mix single-stranded oligos with complementary sequences. The top-strand oligo should include CACC on the 5’ end, while the bottom-strand oligo should include AAAA on the 5’ end.
- For the miRNA vectors, make sure that the top-strand oligo includes TGCT at the 5’ end and that the bottom-strand oligo includes CCTG at the 5’ end.

Answer Id: E10017

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I’m getting no fluorescence signal with my expression clone containing EmGFP. What should I do?

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Answer

Please ensure that the recommended filter sets for detection of fluorescence are used. Use an inverted fluorescence microscope for analysis. If desired, allow the protein expression to continue for 1-3 days before assaying for fluorescence.

Answer Id: E10030

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I’m seeing some basal expression of my shRNA of interest in the absence of tetracycline induction when using the BLOCK-iT™ H1 construct or pLenti4/BLOCK-iT™-DEST construct. What could be causing this?

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Answer

Please check to ensure that your medium containing fetal bovine serum (FBS) is reduced in tetracycline. Many lots of FBS contain tetracycline, as FBS is often isolated from cows that have been fed a diet containing tetracycline, leading to low basal expression of shRNA. Ensure that a cell line expressing the Tet repressor is being used, and that the cells used are transduced at a suitable MOI. If creating your own Tet repressor-expressing cell line, wait at least 24 hours before transducing cells with your shRNA construct.

Answer Id: E10022

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Can I purchase the 5X LR Clonase™ buffer or 5X BP Clonase™ buffer separately?

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Answer

We do not offer the 5X LR Clonase™ buffer and 5X BP Clonase™ buffer as standalone products. They are available as part of the enzyme kits.

Answer Id: E9855

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How do I propagate and maintain the pAd/BLOCK-iT™-DEST vector?

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Answer

We recommend using One Shot™ ccdB Survival T1R chemically competent cells (Cat. No. C751003) for transformation. This strain is resistant to ccdB effects and can support the propagation of plasmids containing the ccdB gene. To maintain integrity for the vector, select for transformants in media containing 50-100 μg/mL ampicillin and 15-30 μg/mL chloramphenicol.

Answer Id: E10014

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What are the packaging limits for lentivirus and adenovirus? Can a 9 kb fragment be packaged into either?

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No, neither lentivirus nor adenovirus can take an insert as large as 9 Kb. Lentiviral packaging limits are around 6 kb and adenoviral packaging limits are around 7-7.5 kb. Above that, no virus is made.

For lentivirus, titers will generally decrease as the size of the insert increases. We have effectively packaged inserts of 5.2 kb with good titer (approx. 0.5 x 10^5 cfu/mL). The size of the wild-type HIV-1 genome is approximately 10 kb. Since the size of the elements required for expression from pLenti vectors add up to approximately 4-4.4 kb, the size of your gene of interest should theoretically not exceed 5.6-6 kb for efficient packaging (see below for packaging limits for individual vectors).
pLenti4/V5-DEST™ vector: 6 kb
pLenti6/V5-DEST™ vector: 6 kb
pLenti6/V5/D-TOPO™ vector: 6 kb
pLenti6/UbC/V5-DEST™ vector: 5.6 kb

For adenovirus, the maximum packagable size is approximately 7-7.5 Kb (see below for packaging limits for individual vectors).
pAd/CMV/V5-DEST™ vector: 6 kb
pAd/PL-DEST™ vector: 7.5 kb

Answer Id: E4095

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