1. Adenoviral vector for gene overexpression
The Vector Core uses the AdenoEasy system from OD260, Inc for our clients. This system requires insertion of your gene into a shuttle vector under control of the CMV promoter. Homologous recombination is then used to incorporate the shuttle vector containing your gene into the adeno-genome in place of the E1 gene region.
In addition to constitutive expression using the CMV promoter, you can also choose inducible expression using a doxycycline-inducible Tet-ON system similar to the one available in our Lentiviral vectors. In the Adenoviral vector Tet-ON, vector core staff eliminated the need for a second vector by replacing the E3 region of the adeno-genome with the the reverse transactivator rtTA. A phage packaging system is used to obtain the recombinant adeno-genome with a 100% recombination rate. We have tested this system using EGFP reporter gene and shown that it gives an almost undetectable basal level in the absence of doxycycline and excellent expression upon induction that increases with the amount of doxycycline used (Fig. 8).
Fig 8.: Second generation doxcycline inducible adenoviral vector expression of EGFP.
2. Adenoviral shRNA mediated adenoviral vector knockdown
The Vector Core staff constructed an AdenoEasy based shRNA vector, which can be used for gene knockdown studies. The hairpin structure is driven by the Polymerase III promoter H1.
3. Detection of replication competent adenoviral vector
The Vector Core uses a PCR-based method approved by the UTHSC IBC for detecting replication competent virus.
Adenoviral Associated Vector
1. Adenoviral-associated vector for gene overexpression and inducible expression
Vector core has a AAV-2 based helper free vector system available. The gene of interest can be subcloned into a shuttle vector pCMV-MCS or pTet-MCS to package the virus in AAV-293 cells.
2. Adenoviral-associated vector for gene knockdown using shRNA or miRNA
Available soon: The vector core is building polymerase III based shRNA and polymerase II based miRNA gene knockdown vectors in the AAV system and will make them available soon.
Lawrence M. Pfeffer, Ph.D.
Interim Vice Chancellor
Office of Research
910 Madison, Suite 608
Memphis, TN 38163
Phone: (901) 448-7125
Fax: (901) 448-7133