Ad-HQ Adenovirus Construction Service
Adenovirus construction is a tedious and time-consuming process. Here at Vector Biolabs, we have developed the Ad-HQ system: a fail-proof construction method that guarantees adenovirus generation. Since 2004, we have successfully constructed tens of thousands of viruses for gene over-expression, shRNA silencing, and gRNA gene editing.
We guarantee all viruses made using our Ad-HQ system will express your GOI with high viral yield and fast delivery.
Step 1: Cloning a Gene of Interest (GOI) into Shuttle Vectors
- Sub-cloning your gene of interest (GOI), shRNA or gRNA into the appropriate, intermediary shuttle/transfer vector.
- Confirmation of vector through restriction digestion.
Step 2: Transfer of Expression Cassette into the Adenovirus Vector
- Transferring the entire expression cassette into the large, adenovirus genome vector.
- Confirmation through restriction mapping and sequencing verification
Step 3: Production of First Generation, Low-Titer Virus Stock
- Linearizing and transfecting the recombinant adenoviral DNA into packaging cells for viral rescue.
- This step takes about approximately 1-2 weeks.
Step 4: Virus Amplification and Concentration
- Amplifying the rescued viruses (primary viral stock) to final of 0.25L of cultured cells.
- Concentrating viruses in 2 ml of viral storage buffer.
Step 5: Titration
- The virus stock is subjected to several rigorous titration procedures including the plaque formation assay. These procedures provide accurate information about the concentration of active adenoviruses in the stock.
- The final viral yield is typically 1-5x10e10 plaque formation unit (PFU)/ml; equivalent to ~10e12 viral particles (VP).
We provide custom services for each step of the adenovirus construction process.
Once completed, you will receive a final viral stock of approximately ~1012 viral particles (1×1010-1×1011 PFU). This process takes ~4-5 weeks to complete.
What you need to provide:
For over-expression adenoviruses, we require a plasmid DNA for the specified gene along with its sequence information and a vector map. Alternatively, if you do not have a plasmid, you can simply send us the sequence or RefSeq# for the GOI.
For shRNA silencing adenoviruses, we require either a working shRNA in plasmid DNA or the sequence of the shRNA you want to use. If you don’t have a working shRNA, you can provide us with the name and species (or RefSeq#) for the gene you want to silence, and we will begin with our shRNA screening/validation process to identify a good shRNA for viral production.
For gRNA adenoviruses, we require a plasmid containing your gRNA cassette or the sequence of the gRNA you want to use.
Quality Control – The AccuTiter Procedure
Accurately titering viral stock is critical to using adenoviruses for gene delivery. Traditionally, the process of determining the active/infectious/”working” viral concentration is tedious and involves numerous steps, each of which can produce wide variations. These potential variations in titration are one of the major causes for discrepancies regarding the MOI used in scientific literature.
We have solved this dilemma with our proprietary titration procedure, AccuTiter, which is guaranteed to give extremely accurate and consistent measurements.
Other Adenoviral Services:
Alongside our adenoviral construction service, we also provide medium (<0.5L of cells) to large scale (1-2 L of culture) virus amplification, 2xCsCl purification, titration, and quality control. If you or a colleague has a virus stock, but aren’t comfortable with the titer, please contact us and we will be happy to assist you.
Need a Custom Virus?We offer a wide range of viral vector services to suit your research needs.
- AAV Production Service
- Adenovirus Amplification Service
- Adenovirus Construction Service
- shRNA Validation Services