Why is it necessary to dilute ligated DNA products before adding them to competent bacterial cells?
Components of the ligation reaction (enzymes, salts) can interfere with transformation, and may reduce the number of recombinant colonies or plaques. We recommend a five-fold dilution of the ligation mix, and adding not more than 1/10 of the diluted volume to the cells. For best results, the volume added should also not exceed 10% of the volume of the competent cells that you are using.
Answer Id: 3098
How can unstable or toxic DNA inserts be maintained in bacteria?
There are a few steps you can take to improve stability of clones with difficult-to-maintain inserts. Supplement the medium with extra nutrients (e.g., add 20-30 mM glucose to Terrific Broth) or try a vector that has a reduced copy number (e.g., pBR322). Some clones can exhibit a high degree of deletions; this is usually a result of the clones having long terminal repeat (LTR) sequences or regions with high secondary structure. To overcome this problem, the cells can be grown at 30°C or ambient temperature (in LB or in a nutrient rich broth like Terrific Broth). Do not to let the cells reach late stationary phase in liquid culture. Alternatively, transform into cells that maintain unstable sequences such as Stbl2™, Stbl3™, or Stbl4™ cells.
Answer Id: 3099
Is S.O.C. medium absolutely required when recovering competent bacterial cells during transformation?
Many media can be used to grow transformed cells, including standard LB, SOB or TB broths. However, S.O.C. is the optimal choice for recovery of the cells before plating. The nutrient-rich formula with added glucose is often important for obtaining maximum transformation efficiencies.
Answer Id: 3100
Can I re-use my competent cells once the tube has been thawed?
Yes, competent cells can be thawed and re-frozen at least once, but be aware that each freeze-thaw cycle can result in up to a 10-fold reduction in transformation efficiency.
To re-freeze unused competent cells, we recommend the following protocol: Pre-cool some new empty vials on ice for 5 min. Thaw the cells, and then aliquot a single-use volume of cells (usually 20-100 ul as recommended in the product manual) into the new tube. Freeze the cells immediately in a dry ice-ethanol bath. (Be sure that ethanol does not leak inside the tube - keep the level of ethanol well below the cap.) Transfer the frozen cells immediately to a -80C freezer, and do not thaw them again until ready for use.
Answer Id: 3103
What are the packaging limits for lentivirus and adenovirus? Can a 9 kb fragment be packaged into either?
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: 4095
How do I know whether to choose lentivirus or adenovirus for viral expression?
If you're interested in stable integration and selection, choose the lentiviral system. We offer both a Directional TOPO® (D-TOPO®) and Gateway® version of the kit to provide flexibility in the cloning of the gene of interest.
If you're looking for transient gene expression, choose the adenoviral system. We offer the Gateway® cloning method for this product. It should be noted, however, that gene expression from both systems is typically detected within 24-48 hours of transduction, so both systems can be used for experiments of a transient nature. The main difference is that lentivirus integrates into the host genome and adenovirus does not. Higher viral titers are achieved with the adenovirus.
Answer Id: 4098
What are the safety issues associated with the use of your viral systems?
Both the lentiviral and adenoviral systems should be used following Biosafety Level 2 (BSL-2). We recommend strict adherence to all CDC guidelines for BSL-2 (as well as institutional guidelines). Life Technologies has also engineered specific safety features into the lentiviral system.
Consult the "Biosafety in Microbiological and Biomedical Laboratories" publication (www.cdc.gov, published by the CDC in the USA, describes BSL-2 handling) and the "Laboratory Biosafety Guidelines" publication (www.phac-aspc.gc.ca, published by the Centre for Emergency Preparedness and Response in Canada) for more information on safe handling of various organisms and the physical requirements for facilities that work with them.
Answer Id: 4099
How should I store lentivirus, adenovirus and 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.
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: 4100
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?
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: 4101
Will I get the same transduction efficiency with both lentivirus and adenovirus in the same cell line?
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: 4102
What is MOI, and how do I know which MOI to use?
MOI stands for multiplicity of infection. Theoretically, an MOI of 1 will provide 1 virus particle for each cell on a plate, while an MOI of 10 represents ten virus particles per cell. However, several factors can influence the optimal MOI including the nature of your mammalian cell line, (non-dividing vs. dividing), transduction efficiency, your application of interest, and your protein of interest.
When transducing your adenoviral or lentiviral construct into the mammalian cell line of choice for the first time, we recommend using a range of MOIs (0. 0.5, 1, 2, 5, 10, 50) to determine the MOI required to obtain optimal gene expression (MOIs greater than 50 (such as MOI 100) are common for the transduction of neurons with lentivirus). After you determine the MOI that gives optimal gene expression, subsequent transductions can be performed at the optimal MOI.
Answer Id: 4103
How does the lentiviral system work? How do I make the lentivirus?
Clone your gene of interest into one of our lentiviral expression vectors. We have a Directional TOPO® version (pLenti6/V5/D-TOPO®) and a Gateway® version (pLenti6/V5-DEST™ vector). Co-transfect your recombinant vector along with the optimized ViraPower™ packaging mix into the 293FT producer cell line using Lipofectamine™ 2000 reagent (if using a different transfection reagent, follow the manufacturer's recommendations). Harvest the viral supernatant and determine the titer of the virus. Add the viral supernatant to your mammalian cell line of interest at the appropriate MOI. Assay for "transient" expression of your recombinant protein or select for stably transduced cells using the appropriate selection antibiotic, if desired, then examine expression of your protein of interest.
Answer Id: 4104
What are the advantages of the lentiviral system?
The ViraPower™ Lentiviral System:
(1) effectively transduces both dividing and non-dividing cells
(2) efficiently delivers the gene of interest to mammalian cells in culture or in vivo
(3) produces a pseudotyped virus with a broadened host range
(4) includes multiple features designed to enhance the biosafety of the system
Answer Id: 4105
What type of virus is the lentivirus?
The lentivirus is a retrovirus and is loosely based on HIV-1, however it has been altered to function solely as a gene delivery vehicle without subsequent viral replication or disease. Specific HIV-1 genes have been deleted to enhance safety. The HIV-1 genes are only expressed in the producer cells (293FT) and none of them are packaged into the viral genome and thus are never expressed in the transduced target cell.
Answer Id: 4106
Can I transduce my specific cell type with a lentivirus? How does lentivirus infect cells?
The lentiviral envelope contains (is pseudotyped with) the vesicular stomatitis virus glycoprotein (VSV-G), which allows the lenitivirus to interact with its target cell in a receptor-independent manner. This receptor-independent entry into the target cell likely involves endocytosis (see Espenshade et al (2002) Proc Natl Acad Sci USA 99:11694; Aiken (1997) J Virol 71:5871). Since the interaction of this pseudotyped lentivirus with mammalian cells is receptor-independent, the lentivirus should be able to (in theory) transduce any mammalian cell type.
Theodore Friedmann was the pioneer in the field of lentiviral host range and has a tremendous number of publications and reviews describing VSV-G pseudotyping of retroviruses and their tropisms:
VSV-G binding to target cells is receptor-independent. It interacts with a common phospholipid (phosphatidylserine has been proposed to be the most probable target) in the target cell membrane and this is how it confers such broad tropism: mammalian cells, fish, etc. (Burns et al (1993) Proc Natl Acad Sci USA 90:8033).
One group has shown that VSV-G pseudotyped retroviruses can transduce amoebas (see Que et. al. Mol. Biochem. Parasitol. (1999) 99:237-45).
The VSV-G element was derived from the San Juan strain of the Indiana serotype (Rose and Gallione (1981) J Virol 39:519), as referenced by Ory et al (1996) PNAS 93:11400).
Answer Id: 4107