It is good virological practice to isolate clones of a recombinant virus, even if the virus was generated from a plasmid or cosmid isolated from a single E. coli clone. A recombinant adenovirus can prove unstable if the expression cassette it carries exceeds the maximum cargo capacity of the vector, or if it encodes a protein that is toxic for the helper cells or interferes with viral replication. Also, some recombinant adenoviruses grow slowly and can rapidly be overcome by RCAs.
We recommend virus plaque isolation especially if you suspect a high level of RCA in your prep, if you have received a virus sample from an unknown third party, if you plan to have your adenovirus vector amplified in a GMP facility, or if you have observed a decrease in transgene expression from your virus lately.
- Isolation of viral clones during virus construction, prior to large-scale virus amplification
- Isolation of viral clones prior to transfer to a GMP facility
- Isolation of clones of a virus that might reveal unstable
- Isolation of clones from a virus suspension that contains replication-competent adenovirus (RCA)
- A purified virus suspension, or a crude virus lysate.
The service includes
- Infection of cell monolayers with a serial dilution of your virus
- Isolation of up to 10 plaques
- Small-scale amplification of each plaque
- Confirmation of virus identity at the DNA level (restriction analysis, PCR, sequencing..), RNA level (RT-PCR..) or protein level (western blot...)
- The crude virus lysates of three clones of your recombinant virus (1 mL per clone)
- A complete report describing the work performed and demonstrating the identity of the viruses we are sending back to you
- 3-4 weeks, depending on the nature of the virus
Important Note on Handling Adenovirus
The use of adenovirus as a genetic vector requires adequate containment equipment and practices. Biosafety Level 2 (BL2) is appropriate for most customers; however, for some, use of BL3 or higher level containment facility may be required. PLEASE NOTE, O.D.260 Inc. will not carry out projects that require BL3 (or above) containment.