 | The Harvard Gene Therapy Initiative Research Vector Core Services |
| The Harvard Gene Therapy Initiative Research Vector Core Services |
The HGTI Research Vector Core provides services to investigators in the production of various viral vectors for research use. The Core lab is currently set up to produce murine leukemia virus (MLV) based retroviral vectors, HIV-1 based lentiviral vectors, adenoviral vectors and adeno-associated viral (AAV) vectors.
The MLV-based viral vectors are usually generated by transient transfection. Depending on the titering method appropriate for the specific vector, virus preparation can usually be completed within 3-6 weeks. The virus yield from the transient transfection supernatant is normally in the high 106 /ml range. VSV-G pseudotyped virus can be further concentrated by ultracentrifuge to 109 /ml. High titer viruses carrying commonly used reporter genes, such as β-galactosidase and green fluorescent protein, are usually available in stocks.
The HIV-based viral vectors are generated by transient transfection. Depending on the titering method appropriate for the specific vector, virus preparation can usually be completed within 3-6 weeks. All virus preparations will be tested and documented to be free of replication competent virus before release. The virus yield from the transient transfection supernatant is normally around 105-106 /ml range. VSV-G pseudotyped virus can be further concentrated by ultracentrifuge to 108 /ml. High titer viruses carrying commonly used reporter genes, such as β-galactosidase and green fluorescent protein, are usually available in stocks.
The adenoviral vectors routinely produced at the core are first generation serotype 5 Ad E1- and E3-deleted vectors. The standard preparation process takes about 5-7 weeks, starting from plasmid transfection, plaque purification, amplification of virus onto 10 15-cm plates of cells and purification of virus by two cycles of CsCl gradient. The yield from a standard preparation is usually about 2-4 ml virus titered between mid 109 to low 1011 pfu/ml.
The recombinant AAV (rAAV) vectors produced at the core are derived from AAV serotype 2. The viruses are generated by transient transfection of the following three plasmids into 293A cells: i) AAV rep/cap expression plasmid, ii) adenovirus miniplasmid and iii) the AAV vector. A standard prep is defined as the virus particles obtained from 10 15-cm plates of transfected cells. The virus is purified by Heparin column chromatography. The final product is dialyzed against PBS and titered by dot blot hybridization. The yield from a standard preparation is usually about 6 ml of virus titered between mid 1011 to mid 1012 particles per ml.
In addition to virus production, the Core also provides plasmid construction services to help investigators to clone their genes of interest into various viral vector plasmids. Depending on the complexity of the cloning procedures, it usually takes about 3-4 weeks (for AAV, MLV, and HIV vectors) to 6-8 weeks (for adenoviral vectors).
| Continue to Virus Request Form. |
Copyright © 2002, Presidents and Trustees of Harvard University. All rights reserved. Site maintained by Adam Lorch, webmaster@hgti.med.harvard.edu.