The NHLBI PEGT Pre-Clinical Grade Vector Production Core at the University of Pittsburgh provides stocks of retroviral and adenoviral pre-clinical grade vectors for gene delivery as well as develops expression pre-clinical grade vectors for use in non viral gene transfer for all NHLBI-supported investigators. In addition, the core provide cell lines, viruses, packaging lines, plasmids, and protocols as needed to NHLBI investigators. The Core also works with the investigators to optimize viral and non-viral gene delivery and gene expression. Furthermore, the Core provides technical assistance and training to individuals in the pre-clinical use of viral and non-viral vectors for gene transfer as needed.

Below are listed the services which can be performed by the PEGT pre-clinical Vector Core:

1. To provide stocks of previously generated viral and non viral vectors. The Core will provide NHLBI-supported investigators with previously generated recombinant retroviral, adenoviral and plasmid-based vectors.

The inventory is available for viewing.

2. Construction of recombinant plasmid vector. The core will insert the appropriate cDNA into one of a number of different eucaryotic expression vectors. The cloning junctions will be confirmed by DNA sequencing. The investigator will be provided with 0.5 to 1.0 mg of clean plasmid.

3. Construction of a recombinant adenoviral vector: The Core will construct an adenoviral shuttle plasmid expressing the appropriate gene. The cloning junctions will be confirmed by DNA sequencing. A first generation adenoviral vector will be constructed by co-transfection into 293-based CRE8 cells and the resulting plaques isolated and tested.

4. Generation of a high titer stock of an adenoviral vector. A stock of recombinant adenovirus will be amplified in 293 cells and tested for replication competent adenovirus. The investigator will receive at least 1011 viral particles.

5. Construction of a retroviral vector. The core will insert the appropriate cDNA into one of a number of different retroviral vectors. The cloning junctions will be validated by DNA sequencing.

6. Generation of retroviral supernatant. The recombinant retroviral vector will be transfected into the appropriate ecotropic, amphotropic, or pseudotyped packaging cell line and retroviral supernatant collected. The investigator will receive at least 6 ml of retroviral supernatant.

For questions about custom or typical services offered by the NHLBI PEGT pre-clinical Vector Core, please e-mail Andrea Gambotto, M.D., Human Gene Therapy Center, University of Pittsburgh, Pittsburgh PA at: mailto:agamb@pitt.edu

To apply for services, please download the application form (requires Adobe Acrobat reader) and fax to 212-746-8824. An online form will soon be available. Applications will be reviewed on a rolling basis.