Typically, the Core provides a shuttle plasmid to the investigator who inserts the cDNA of interest to construct a viral vector. Recombinant viral stocks generated from viral plaques are provided to the investigator to evaluate gene expression using the appropriate methodology (either structural or functional assay). Retroviral and lentiviral vectors and packaging cell lines are also available from the Core. Transient transfection using a three-plasmid system is used to produce high titer retroviral and lentiviral supernatants from 293T cells that can be pseudotyped if desired. The Core also supports the use of non-viral vectors for gene transfer by maintaining a repository. Included is the Ultimate™ ORF Lite library from Life Technologies Inc.
A series of plasmid-based expression vectors containing marker genes, cytokines or polylinkers that were originally developed under the National Gene Vector Laboratory program are also available to investigators.
Please contact the Director for scientific advice and availability of services, pricing, details and scheduling of services.
List of services
Typically, the Core provides a viral shuttle plasmid to the investigator who inserts the cDNA of interest. Before the vector is amplified and titered, the Core provides the investigator with recombinant viral stocks generated from viral plaques to evaluate gene expression using the appropriate methodology (either structural or functional assay).
- Co-transfection of adenoviral shuttle vectors with adenovirus helper DNA into producer cell lines
- Plaque purification of viral clones
- Growth and purification of high titer recombinant stocks
- Assay of recombinant and wild type virus titers by plaque assay or TaqMan PCR
- E1-replacement; E1, E3 deleted; E1,E3,E4 deleted; E1,E3-deleted ts125 second-generation vectors
- Targeted Ad vectors (fiber modified)
- Tet-ON adenovirus vectors (single vector, E1/E3 replacement)
- Stock vectors (marker viruses expressing gfp, luciferase or b-galactosidase and empty vectors) or custom constructs
The Core uses transient transfection, using a three-plasmid system, to produce high titer retroviral supernatants from 293T cells that can be pseudotyped if desired.
- Retrovirus (by transient transfection)
- Lentivirus (by transient transfection)
- Amplification/propagation of desired volume
- Titration (ELISA, colony forming assay, TaqMan qRT-PCR)
- Quality control (identity testing, functional testing, RCA testing)
The Core also supports the use of non-viral vectors for gene transfer by maintaining a repository. A series of plasmid-based vectors containing marker genes, cytokines or polylinkers that were originally developed under the National Gene Vector Laboratory program are available to investigators following inquiry with the Director. The repository also includes the Ultimate™ ORF Lite human cDNA collection from Life Technologies. Ultimate™ ORFs are provided in a Gateway® entry vector.This 16,740 clone collection is maintained as frozen glycerol stocks in 96-well plates at -80°C, and is provided as live starter cultures in LB-Kan medium to investigators.
- Stock vectors
- Construction of new vectors
- Propagation (0.05-10L volumes)
- Purification (Qiagen, CsCl banding or column chromatography)
- Quality control (testing for endotoxin, protein, RNA and E. coli DNA contamination)
- Provide viral cloning plasmids for adenovirus, lentivirus and retrovirus vectors
- GLP manufacturing capability
Ultimate™ ORF clone collection from Life Technologies
- Individual clones and functional clone collections