Retroviral and Lentiviral Vector Production Details
Transfection
Retroviral or lentiviral vector is generated using a standard transient three-plasmid (for retrovirus) or four-plasmid (for lentivirus) transfection in 293T or Phoenix-gp cells based on investigator-provided vector plasmid. Transiently produced lentivirus generated using a four-plasmid system is considered a 3rd generation lentiviral vector. For transfection, the Vector Core provides the necessary packaging plasmids including the MLV or lenti-gag/pol, envelope and Rev plasmids (as applicable). Available envelopes include the ecotropic envelope, amphotropic envelope, gibbon ape leukemia virus (GALV) envelope, feline endogenous virus envelope (RD114), and the VSV-G envelope.
Stable Producer Cell Lines
The VVC is able to generate stable producer lines of the vector of choice pseudotyped with either the GALV or RD114 envelope (retrovirus only at this time). This option is recommended for investigators that require larger amounts of vector for large scale studies (>1 Liter), for investigators who expect to need a consistent quality vector supernate over an extended period of time, or for instances where verification of vector integrity is critical. The VVC recommends that proof of principle be obtained using a transient vector before requesting a stable producer. Generation of a stable producer clone takes several months to be completed. After a research cell bank has been prepared (20 vials at 3 x 106 cells/vial), vector supernate can be harvested from the cells at a fraction of the price as compared to transfection.
Plasmids
Packaging plasmids necessary for transfection are provided by the Vector Core. The identity of each plasmid has been verified and purity of each preparation has been checked. Sequences or maps of most plasmids are available upon request. The Vector Core is able to purify vector plasmid provided by the investigator at two grades: Technical Grade (100-500 µg yield prepared using Qiagen kits) or Research Grade (10 mg commercially prepared).
Vector Titration
The Vector Core provides vector titration services using PCR or flow cytometry depending on the presence of a fluorescent protein. Briefly, mouse NIH 3T3 or human HT1080 cells (as applicable) are infected for 4 hours with limiting dilutions of vector supernate in the presence of polybrene and analyzed for expression of fluorescent protein or detection of vector or packaging sequences by PCR. The titer represents the relative infectivity of the vector as measured on the target cell of choice and is expressed as Infectious Units (IU)/mL. Different target cells, or different infection protocols, may yield different results.
Concentration
Vector supernate can be concentrated using ultrafiltration. Recoveries (65-90%) vary depending on the envelope used and stability of the viral particle. This technique does not separate empty from functional viral particles. Therefore, at very low titers, concentration may not always yield an improved titer due to the presence of inhibitory empty viral particles.
Quality Control Testing
Non-GMP Quality Control (QC) testing includes Mycoplasma testing (PCR); Sterility Testing (14-day assay for aerobic and anaerobic bacteria and fungi according to the USP); RCR Testing (PCR or Marker Rescue); and RCL Testing (PCR or p24 ELISA).
Contact Us
For further information regarding the Viral Vector Core, please contact Dr. Han van der Loo, Director, Vector Production Facility at 513-475-4093 (Ext. 1). For further information about the Division of Experimental Hematology and Cancer Biology , please contact Dr. Yi Zheng at 513-636-0364. The Division of Experimental Hematology and Cancer Biology can be found in Room 7.205 of Location S (Research Foundation Building).
