Q. What is the required biosafety level for handling recombinant adenovirus?
A. Our recombinant adenoviruses that are deleted in the E1 and E3 regions will replicate in HEK293 cells, but not in other cell lines. According to the NIH Office of Biosafety, recombinant human adenovirus has been classified in biosafety level II for agents considered of ordinary potential harm, and you need BL-2 level facility to work with it. It should be noted that cell culture facilities in most institutes are certified as BL-2 level.
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 http://bmbl.od.nih.gov.
Q. What type of cells are used to produce and grow recombinant Adenoviruses in your systems?
A. Human Embryonic Kidney cell line, the HEK-293 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.
Q. How are recombinant adenoviruses purified and virus titers determined?
A. The amplified recombinant adenovirus was purified on 2 sequential cesium chloride gradients and then dialyzed with a buffer (PBS, 10% glycerol, pH7.4) to reduce the salt concentration. The titer of purified virus can be determined with (1) OD260 Assay or (2) Plaque Assay.
The OD260 assay measures the concentration of viral DNA and protein. It does not distinguish between intact, infectious viruses and damaged, non-infectious viruses. It is a physical assay measuring the concentration of total viruses, live and dead. Based on OD260 data, the concentration of viral particles (VP) can be calculated using the following formula:
Adenovirus titer in viral particles/ml = OD value x d x 1.1 x 1012; ( d= dilution factor)
The plaque assay measures the concentration of infectious viruses, and therefore it is a biological assay. Basically, a mono-layer of HEK293 cells are infected with a series of virus dilutions. Viruses will propagate in infected cells, and eventually leading to the formation of holes or plaques. Thus, a PFU can be determined from a plaque assay.
Q. What are the differences between viral particle (VP) and plaque formation unit (PFU)?
A. Viral particles (VPs) represent the total number of viral particles (live and dead combined). Because of variations in viral preparations the ratio of live/dead varies significantly. Thus, VP does not reflect the amount of live virus in the preparation.
PFU (plaque formation unit) represents the number of infectious or live viruses. It reflects the amount of working viruses in the preparation.
For most virus preps, the VP/PFU ratio is 20:1 to 50:1.
Q. What are the conditions recommended for the storage of recombinant adenovirus preparations?
A. For long-term storage, the virus should be stored at -800°C, especially after CsCl purification. At -800C, the virus could be stable for at least a year. However, the repeated freeze-and-thaw should be avoided, since it will cause significant decrease of titer.
Q. What size of insert can be cloned into the Adenoviral expression system?
A. The insert size limitation for the DE1/E3 deleted Ad5 vector is about 8 Kb in length.
Q. What are RCAs?
A. The replication competent adenoviruses (RCAs) is a result of the rare double crossover through overlapping sequences present in the recombinant adenovirus and the genome of HEK293 cells. This event results in the replacement of the transgene by E1 region. Once this happens, the adenovirus could replicate, without the need of a complementing cell line. Usually two methods, non-complementary A549 cells or PCR could be used to detect RCA in a recombinant adenoviral preparation.
According to NIH guideline, <1 plaque in about 1E4 viruses is considered safe to use. To avoid the occurrence of RCA, viruses should be produced and amplified in low passage packaging cells.
Q. What MOI (multiplicity of infection) should I use with my cells?
A. Usually, a MOI of 1 is suitable to infect 293 cells. If you use other cell lines and the susceptibility to Adenovirus infection is unknown, you should test a MOI (number of virus per cell) range between 1 and 1000. The MOI may be increased up to 10000 when testing a mouse cell line. For most cell lines, the transfer of a reporter gene (for example, GFP) to 100% of cells, without any signs of toxicity, can be achieved with a MOI of 10-100.
Q. Which viral expression system (adenovirus, retrovirus and lentivirus) should I use for my experiments?
A. Adenovirus can infect many types of mammalian including dividing and non-dividing or primary cells with high efficiency. They do not disrupt the genome of the host cell but remain in the nucleus as episomal DNA. However, adenovirus only provides a transient, high level gene expression in vitro and in vivo.
Retrovirus has low infection efficiency (<30%) in most cell types, and requires active cell division. In addition, there is a significant risk of integration into the host genome, leading to mutation of genes or activation of onco-genes in the host system. Unlike adenovirus, the costs of production of recombinant retrovirus are very expensive, which may limit your in vivo application.
Like Retrovirus, Lentivirus has low infection efficiency (<30%) in both dividing and non-dividing cells. Lentivirus also can integrate into the host genome, leading to mutation of genes or activation of oncogenes in the host system. Like retrovirus, the costs of production of recombinant lentiviruses are expensive.
Q: What’s the Biosafety requirement for using AAV
A: Adeno-Associated Virus (AAV) requires Biosafety Level 2 (BSL-2) practices and procedures. 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 http://bmbl.od.nih.gov.
Q: What is recombinant AAV (rAAV)?
A: Recombinant AAV is the artificial AAV which does not contain any AAV rep and cap genes which encode viral replication and structural proteins, respectively. The rep and cap are replaced with a gene or construct of interest which is flanked by the ITRs which contain all the cis-acting elements necessary for replication and packaging. Though AAV possesses a 4.7 kb genome, efficient packaging of rAAV can be performed with constructs ranging from 4.1 kb to 4.9 kb in size.
Q: Is recombinant AAV replication deficient?
A: Because AAV is a replication defective virus, its replication depend on other virus such as adenovirus and herps virus. these virus also called helper virus. Studies identified AAV only need some of Helper virus products (E1A and E1B,, E2A, E4 and VA) which are provided in trans by another vector or by stably expression cell line.
Q: Is the recombinant AAV safe?
A: The recombinant AAV (rAAV) composed by several plasmids (cis plasmid, Helper plasmid, rep/Cap plasmid). Cis plasmid and Helper do not share any regions of homology with the rep/cap-gene containing plasmid , preventing the production of wild-type AAV-2 through recombination system. AAV is not reported to link to any human disease.
Q: Which serotype should I use in my experiment?
A: AAV(s) has been used for in vivo studies extensively. However data regarding which serotype and dose are best for each application/tissue are not clear, and sometimes even controversial. This is probably due to the different experimental procedures used and end-point readout.
- Below are some preliminary guidelines based on literatures.
- AAV1: CNS, Eye, Heart, Lung, Skeletal muscle
- AAV2: CNS, Eye
- AAV5: CNS, Eye, Lung
- AAV6: Adipose, Heart, Liver, Lung, Skeletal muscle
- AAV8: Adipose, CNS, Eye, Liver, Skeletal muscle;
- AAV9: Adipose, CNS, Eye, Heart, Liver, Lung, Skeletal muscle
Q: How to store recombinant AAV?
A: Stability studies show that purified rAAV are highly stable at temperatures of 4°C or less. However, it is advisable to be aliquoted upon receipt of rAAV and be stored at -80°C. Once an aliquot is thawed it can be stored at 4°C for up two weeks. Stability studies show rAAV vectors are highly stable at temperatures of 4°C or less. Avoiding repeated thawing and freezing.
Q: What’s the difference between physical and genomic particles?
A: The rAAV particle that have been successfully packaged with the genome are called genomic particles which has the ability to transduce the cells they come into contact with and are therefore functional. During the AAV packaging process many particles are formed lacking the genomic DNA, which lack the ability to transduce the cells they come into contact with and are therefore non-functional.