Creation of Ad Vector
With this service, you will receive the pShutlleX transfer vector to clone your transgene. Please contact us to determine which transfer vector to use and which Adenovirus backbone is most appropriate for your application (we do not provide aliquots of pAdenoX or pAd5F35). We ask that you send us 100 ug of the shuttle vector containing your transgene. Once we receive the shuttle vector, we will move your transgene into the pAdenoX (Clontech now Takara Bio) or into the pAd5F35 vector and prepare the new construct for transfection. Following transfection, 6-8 discrete plaques will be selected and undergo small expansion. One-third of the small expansions will be sent to you for testing transgene expression. One-third of the small expansions will be used to extract and analyze viral DNAs if necessary. The final one-third will be stored until you inform us of which isolate to expand. View an outline of an Adenovirus Creation, Expansion and Purification.
Transfection and Plaque Isolation (Ad Vector)
With this service, you do all the cloning. Once you have inserted your transgene into one of the Ad plasmid vectors, you send us 100 ug of the final plasmid and we prepare your construct for transfection. Following transfection, 6-8 discrete plaques will be selected and undergo small expansion. One-third of the small expansions will be sent to you for testing transgene expression. One-third of the small expansions will be used to extract and analyze viral DNAs if necessary. The final one-third will be stored until you inform us of which isolate to expand.
Small-Expansion (Ad Vector)
We have found some investigators wishing to expand an Ad vector which they obtained from a third party often have minimal information about the vector titer, purification state, age, freeze/thaw history, etc. This precludes us from beginning with the large-scale expansion. To circumvent this problem, we begin by infecting one 60mm dish of 293 cells with the virus. We collect virus and use it to do a medium-scale infection (a.k.a. Preparation of lysate).
Preparation of lysate (Ad Vector)
This service is most commonly used when we have created a custom virus. Crude virus from the initial plaque expansion is used to infect five, 150 mm dishes of 293 cells. The lysate from this expansion is used to infect the cells for the Large-Scale Expansion. This service is also used when investigators want an adenovirus expanded on the large-scale but do not have the 1x10e12 particles needed for infection.
Large-Scale Expansion and Purification (Ad Vector)
The Large-Scale Expansion is the final expansion in the production of an adenovirus. The infection begins with either 1x10e12 particles of purified virus or crude viral lysate collected from the Preparation of lysate. The crude virus collected from the cell factory is purified using cesium chloride ultracentrifugation. The purified virus is desalted and diluted in a specific storage buffer to 5x10e12 particles/ml.
Quality Control Services
Adenovirus Infectious Titer – Rapid Assay
We use the AdenoX Rapid Titer kit (Clontech now Takara Bio) for this service. This immunohistochemical assay involves making 3 serials dilutions of the virus and infecting 6 wells of a 24-well plate of 293 cells. At 48 hours post-infection, the cells are fixed and immunostained with an anti-hexon antibody. Individual or clusters of stained cells are counted, and the resulting figures are applied to a formula that will give the infectious titer/ml. This protocol takes 3 days to complete.
Adenovirus Infectious Titer – Plaque Assay
The standard protocol involves making 6 serial dilutions of the virus and infecting two, 6-well plates of 293 cells with the virus. At ~16 hours post-infection, the cultures are overlaid with an agar/media mixture. The cells are observed daily for the formation of plaques. The plaques are counted and then applied to a formula that will give the infectious titer/ml. The protocol takes 10-14 days to complete.
Assay for Replication Competent Adenovirus (RCA)
Preparations are judged to be free of Replication Competent Adenovirus (marked as negative) if they pass a test of incubation of 10e9 particles of recombinant Adenovirus on A549 cells for 11 days. Appropriate positive and negative controls are run with each RCA test.
Test for Sterility
Sterility testing is performed using Trypticase Soy Broth for detection of yeast, mold, and aerobic bacteria and Fluid Thioglycolate medium for detection of yeast, mold, aerobic and facultative anaerobic bacteria. Preparations are judged to be sterile (marked as negative) if they pass the test of incubation for 14 days.
Test for Endotoxin
Endotoxin testing is performed using a quantitative kinetic-chromogenic Limulus Amebocyte Lysate or LAL test.
Test for Mycoplasma
Cell substrates for vector production (e.g. 293 cells) are routinely tested for mycoplasma (MycoAlert™, Pierce). We can also test virus preparations for mycoplasma contamination.