 |
|
|
FAQ: Adeno-Quest 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 subtype of the Ad5 viral backbone used for the Adeno-Quest constructs? How strong is the Ad MLP (Major Late Promoter), or BM5 promoter, compared to the CMV5 promoter?
Q. Is the adenovirus safe to handle? A. The recombinant adenoviruses made with our Adeno-Quest™ 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 (by CsCl usually). 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. Reference:
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 x 1013 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: 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 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: Jani, A., H. Lochmuller, et al. (1997). "Generation, validation, and large scale production of adenoviral recombinants with large size inserts such as a 6.3 kb human dystrophin cDNA." J Virol Methods 64(2): 111-24.
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. Our subclone is a very reliable post-crisis cell line that adheres strongly to plastic, grows very easily, and performs very well in plaque assays and in transfection protocols. 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. 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.
Q. What is the subtype of the Ad5 viral backbone used for theAdeno-Quest™ constructs? A. The viral backbone we use for the Adeno-Quest™ constructs is an Ad5 subtype delta L327, deleted in a XbaI fragment of 1.89 kb that covers almost all of the E3 region from 28,593-30,471 (78.5-84.3 Map units). The backbone is further E1 deleted from nucleotides 358-3,332 (numbers are based on Ad5 wt sequence, map units 1.4 to 9.4). This removes nearly all the E1 sequences between the viral packaging domain and the coding sequence for pIX.
Q. How strong is the Ad MLP (Major Late Promoter), or BM5 promoter, compared to the CMV5 promoter? A. Although the MLP is active in the early phase of the infection, its transcriptional activity is increased 30-50 fold during the late phase i.e. in 293 cells only. In non-permissive cells, its strength compares with a minimal CMV promoter.
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 pQBI-AdCMV5-IRES-GFP, 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). 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. |
|||||||||||||||||||||||||||||||
|
||||||||||||||||||||||||||||||||
|
| . |
