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FAQ: AdenoVator Is the adenovirus safe to handle? What are RCAs and how can I avoid them? For in vivo use (animal models), is the cesium chloride purification required? What are the conditions recommended for the storage of recombinant Adenovirus preparations? Do I risk losing titer after freezing and thawing my viral preparation? What type of buffer is recommended for dialysis? What is the capacity of cloning into the Adenovirus as an expression system? Why are there only 1-2 cloning sites after the CMV5 promoter in some of your transfer vectors? What type of cells are used to produce and grow recombinant Adenoviruses in your systems? What is the deletion in the E1 sequence in your AdenoVator kits? What is the difference between the CMV and the CMV5 promoters? What is the best way to screen for recombinant DNA made with the AdenoVator kit?
Q. Is the adenovirus safe to handle? A. The recombinant adenoviruses made with our expression system kits are defective viruses that are deleted in the E1 and E3 regions; they will not replicate in cells other than complementing cells (293 cells). According to references issued by the NIH (National Institute of Health) Office of Biosafety, U.S. Department of Health, all serotypes of human adenoviruses have been classified in biosafety level II. Level II consists of agents that are to be considered of ordinary potential harm. For more information on biosafety levels, please refer to the following CDC publication: Biosafety in Microbiological and Biomedical Laboratories, 4th Edition, May 1999; this publication is also available at
Q. What are RCAs and how can I avoid them? A. One concern when working with Adenoviral vectors is the possible occurrence of replication competent adenoviruses (RCAs) in a population of replication deficient adenoviruses (Ad). RCAs can emerge as a result of a double crossover event between the homologous overlapping sequences present in the recombinant Ad and the 293 genome (Lochmüller, 1994). This event results in the loss of the transgene and its replacement by the E1 region (Zhu, 1999) thus rendering the Ad replication competent without the need of a complementing cell line. Lochmüller et al. demonstrated that Ad stocks contained an increasing amount of RCAs after increasing number of passages in 293 cells. To avoid the emergence of E1-containing Ad, it is necessary to 1- always plaque purify the adenoviral stocks to be amplified, and 2- keep the passage number in 293 cells as low as possible, ideally no higher than 4. Included below is a list of references on occurrence, characterization and screening of RCAs in adenoviral stocks. Hehir, K. M., D. Armentano, et al. (1996). "Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence." J Virol 70(12): 8459-67. Lochmuller, H., A. Jani, et al. (1994). "Emergence of early region 1-containing replication-competent adenovirus in stocks of replication-defective adenovirus recombinants (delta E1 delta E3) during multiple passages in 293 cells." Hum Gene Ther 5(12): 1485-91. Louis, N., C. Evelegh, et al. (1997). "Cloning and sequencing of the cellular-viral junctions from the human adenovirus type 5 transformed 293 cell line." Virology 233(2): 423-9. Zhang, W.-w. K., Patricia E.; Roth, Jack A. (1995). "Detection Of Wild-type Contamination In A Recombinant Adenoviral Preparation by PCR." BioTechniques 18(3): 444-447. Zhu, J., M. Grace, et al. (1999). "Characterization of replication-competent adenovirus isolates from large-scale production of a recombinant adenoviral vector." Hum Gene Ther 10(1): 113-21.
Q. For in vivo use (animal models), is the cesium chloride purification required? A. Yes: CsCl purification is essential in order to 1) remove defective particles, 2) remove condition media with its contaminants from the viral preparation, 3) concentrate the virus to a level suitable for injection and 4) resuspend the virus in a buffer suitable for injection. For in vivo work, the virus must be purified since a cell lysate contains defective particles, a large quantity of fiber and penton proteins which are known to be cytotoxic, as well as media, serum and cellular debris. If the virus is to be injected into animals, the presence of these components will elicit a very strong immune response.
Q. What are the conditions recommended for the storage of recombinant Adenovirus preparations? A. The viruses should be stored -80°C especially after purification from culture media. In optimal buffer (10mM Tris HCl pH 8.0, 2 mM MgCl2, 4% sucrose) the virus will be stable for 1-2 years; the virus should also be aliquoted to avoid multiple freeze-thaw cycles. Long-term storage at -20°C is not recommended. Virus is DMEM supplemented with serum needs to be stored the same way as purified particles but is usually much more stable than in buffer. Storage of virus in DMEM with serum at 4°C for up to 1 week to avoid freeze-thaw cycles is acceptable.
Q. Do I risk losing titer after freezing and thawing my viral preparation? A. Virus in DMEM can endure repeated (more than 30) freezing/thawing cycles without any drop in titer. In the case of CsCl purified virus, a drop of up to 1 log in titer can be observed after storage at -80°C depending on the buffer used. The buffer and pH in particular are critical for preservation of the titer. For optimal stability upon storage, we recommend the following buffer: Tris 10mM, pH 8.0, with 2 mM MgCl2 and 4% sucrose (Nyberg-Hoffman, C. and E. Aguilar-Cordova (1999). "Instability of adenoviral vectors during transport and its implication for clinical studies." Nat Med 5(8): 955-7.
Q. What type of buffer is recommended for dialysis? A. The best buffer to use is 10 mM Tris pH 8.0, 2mM MgCl2, 4% sucrose; this buffer enables one to concentrate the virus to approximately 1 x1013 VP/mL (viral particle/mL) without precipitation and provides very good stability for long-term storage and shipping. If the virus is to be used for animal studies, a buffer with glycerol should not be used since it is difficult to inject. PBS buffers can also be used but do not provide very good viral stability and should be avoided if the virus has to be concentrated; the particles will likely precipitate due to the low pH (~7) involved. Using a PBS buffer will enable concentration of the virus up to approximately 5 x 1011 VP/mL without precipitation. Viruses in PBS buffer will also be severely affected by repeated freeze/thaw cycles. For these reasons we recommend the Tris buffer over PBS for all applications. Reference:
Q. What is the capacity of cloning into the Adenovirus as an expression system? A. The cloning capacity is the maximal length of recombinant DNA that can produce infectious viral particles without reduction in the efficiency of viral replication, and corresponds to 105% of the wild type genome. The maximal cloning capacity on the other hand is the maximal length of recombinant DNA that can allow production of a recombinant virus. A recombinant virus without gene rearrangement is however difficult to obtain under these conditions. It was demonstrated that the maximum size of DNA that can be packaged is 106.5% of the wild type virus. However the virus produces plaques that are smaller and appear more slowly then usual. The yield of production is reduced 2 - 10 fold compared to a virus that would have 104 - 105 % of the wild type. For these reasons it is strongly recommended not to exceed a DNA length of 105% of the wild type. Reference:
Q. Will extra bases in the 5' and 3' UTRs (Untranslated regions) of my gene cloned into a transfer vector affect protein expression? A. For optimal protein expression, UTRs have to be as small as possible, especially in 5' to avoid formation of secondary structures in the mRNA. A minimal length (7-8 bases) in front of the ATG is acceptable, especially if the Kozak consensus sequence (GCCGCCACCATGG) is included. There are no special requirements for the 3'UTR.
Q. Why are there only 1-2 cloning sites after the CMV5 promoter in some of your transfer vectors? A. The powerful CMV5 promoter available in some of our transfer vectors is optimal for the overexpression of proteins. In these vectors, only one or two cloning sites are available instead of a multiple cloning site (MCS) for a good reason: in order to achieve high expression levels, the number of untranslated bases between the promoter and the start codon must be minimal and it is recommended to avoid adding untranslated bases in 5' (i.e. a MCS) as much as possible when cloning in order to obtain optimal expression.
Q. What type of cells are used to produce and grow recombinant Adenoviruses in your systems? A.
The cells used in the kit for the production of recombinant Adenoviruses
are a Human Embryonic Kidney cell line, the HEK-293A cells (A stands for
adherent cells). The 293 cells contain the full E1 region of the Adenovirus
type 5, from nucleotides 1 to 4355 of Ad5 wt, making these cells suitable
for the generation and growth of helper-independent recombinant Adenoviruses.
References
: Louis, N., C. Evelegh, et al. (1997). "Cloning and sequencing of the cellular-viral junctions from the human adenovirus type 5 transformed 293 cell line." Virology 233(2): 423-9.
Q. What is the deletion in the E1 sequence in your AdenoVatorTM kit? A. The E1 deletion in the pAdenoVator E1/E3 vector is from nucleotides 1 to 3,533. The final virus made with the AdenoVatorTM system also contains nucleotides 1-480 provided by the transfer vector. The Ad sequences inserted in the HEK-293 cells are from nucleotide 1 to 4,344 (Ad5 wt sequences).
Q. What is the difference between the CMV and the CMV5 promoters? A. The CMV5 is an enhanced version of the CMV promoter. The CMV5 promoter in our new AdenoVatorTM vectors was constructed by the insertion, downstream of the transcription start site of the human CMV IE promoter-enhancer, of the adenovirus tripartite leader (Ad-tpl) with the adenovirus major late enhancer bracketed by splice donor and acceptor sites. The Ad-tpl binds translation-initiating proteins much more efficiently than most messages. Viruses with CMV5 express about 6-10 folds more protein in 293 cells than viruses with CMV. Reference:
Q. Is it possible to first transform the BJ5183 with pAdenoVator E1/E3 and then make this recombinant strain electrocompetent for transformation with the linearized transfer vector? A. No, it is not possible. Plasmids grown in BJ5183 are very susceptible to rearrangements. Growth and amplification of plasmids should not be performed in that strain of bacteria. That is why the DH5 strain is also included in the AdenoVatorTM kit, it allows for amplification without rearrangements (does not support recombination).
Q. What is the best way to screen for recombinant DNA made with the AdenoVatorTM kit? A. The restriction enzyme Bst XI gives a very good diagnostic pattern to screen recombinants with the AdenovatorTM system. The restriction fragments of the pAdenoVator-1 plasmid are as follows: 1- 11921
bp Fragment number 2 is the only one that will vary with the size of insert and from one transfer vector to another. With pAdenoVator-CMV5, it shifts up to about 11-12 kb (and higher with the insert) whereas with pShuttle, it shifts to approximately 10.5 kb. This band may sometimes be confused with band 1 but disappearance of band 2 is a good indication of positive recombinant. With pAdenoVator-CMV5-IRES-GFP/BFP, band 2 is cut in the IRES fragment to give a smaller shift of that band and an additional band at approximately 2 kb. This, in conjunction with the Pac I restriction pattern, makes identification of positive recombinants very straightforward.
Q. Upon screening recombinants with Pac I, some clones give a 30-35 kb band and a 3.0 kb band, others the same 30-35 kb and a 4.5 kb band. Which one is the good pattern? A. Some recombinants might come from a recombination event between the right side homologous regions and the origins of replication instead of with the left homologous regions (see applications manual). These 2 recombinants are equally good and can very well be used but will give different restriction patterns. In our own experience, the pattern giving a band at 4.5 kb is generally the most abundant.
Q. If I have a virus expressing the GFP or the BFP, how soon after infection can I start seeing some fluorescence? A. Infected cells should start expressing a detectable level of GFP or BFP 8-20 hours after infection. The level of expression will vary with the promoter used and the cell type so in some instances it may take up to 24 hours.
A. An IRES, or Internal Ribosomal Entry Segment, is a sequence that supports translation initiation from the second cistron in a dicistronic message. In our transfer vector pAdenoVator-CMV5-IRES-GFP or BFP, the first cistron is gene X (i.e. your favorite gene) and the second cistron is the GFP. IRES elements were first identified from the encephalomyocarditis virus messages (Ghattas et al., (1991) Mol. Cell. Biol. 11:5848-5859) but were later found in some eukaryotic messages. The secondary structure of the IRES appears to be very important for its function, more important than the sequence itself. Note that expression levels of the two genes in this dicistronic message is not equimolar; from our own experience we estimate the proportion of genes expressed to be 3:1 (1 being the gene under IRES i.e. the GFP or BFP in our construct). See reference: Mosser, D. D., A. W. Caron, et al. (1997). "Use of a dicistronic expression cassette encoding the green fluorescent protein for the screening and selection of cells expressing inducible gene products." Biotechniques 22(1): 150-61. |
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