In contrast to many gene transfer vector cores around the country, which tend to provide very standard gene transfer vectors and limited consultative services, the Research Vector Core of the DF/HCC was established in 1998 with the specific mission to provide state-of-the-art gene transfer technology and expert advice to DF/HCC investigators. The DF/HCC Vector Core is closely allied with investigators at the Harvard Gene Therapy Initiative who are focused on the development of new gene therapy vectors and associated technologies, so that as new advances in the area of vector development are made, they can be rapidly offered to Core users. Over the past several years, a number of new gene transfer tools have been developed by HGTI investigators and subsequently introduced into the Vector Core 'portfolio' of products/service. As a result, the Vector Core has attracted a lot of interest, receiving and processing over 250 virus requests each year. The current portfolio of products/services includes:
1. Murine leukemia virus (MLV) based retroviral vectors:
A. the vectors: Cloning plasmids are available to express the gene(s) of interest as the single gene product from the vector, or to insert them upstream of IRES-GFP, which provides a convenient way of monitoring infected cells. The gene products can be expressed from the regular MLV LTR promoter, or a number of internal promoters including CMV, PGK, EF1a and Ubc. The core also has successful experience in producing virus that contains special gene regulation cassettes, such as siRNA.
B. virus production: Viruses are generated by three plasmid cotransfection (MLV gag-pol, Env, and the vector plasmid) into 293T cells by transient transfection. The method allows for great flexibility in that vectors can be made with a variety of different envelope proteins, including VSV-G, amphotropic Env, ecotropic Env, GalvEnv, RD114 and 10A1 for different host ranges and transduction efficiency.
Virus production is performed in units of "standard preps". A standard prep of retrovirus is defined as the amount of virus generated from 20 10cm plates of transfected cells. The yield from such a scale of production is usually in the range of low to mid 10^8 infectious units. Viral supernatants are collected twice after transfection, filtered through 0.45 um member, and either frozen down (-80oC) in aliquots, or concentrated to smaller volume, depending on the Env proteins and investigator's preference. A small aliquot is used to titer the virus either by FACS analysis on infected cells (for those expressing GFP) or by Southern blot analysis.
2. Human Immunodeficiency Virus (HIV) based lentiviral vectors:
A. the vectors: Cloning plasmids are available to express the gene(s) of interest as the single gene product from the vector, or to insert them upstream of IRES-GFP, which provides a convenient way of monitoring infected cells. The gene products can be expressed from a number of internal promoters including CMV, PGK, EF1a and MPSV. The core also has successful experience in producing virus that contains special gene regulation cassettes, such as antisense RNA.
B. virus production: Viruses are generated by five plasmid cotransfection (HIV gag-pol, rev, tat, Env and the vector plasmid) into 293T cells by transient transfection. The method allows for great flexibility in that vectors can be made with a variety of different envelope proteins, including VSV-G, amphotropic Env, ecotropic Env, and 10A1 for different host ranges and transduction efficiency.
Virus production is performed in units of "standard preps". A standard prep of retrovirus is defined as the amount of virus generated from 20 10cm plates of transfected cells. The yield from such a scale of production is usually in the range of low to mid 10^8 infectious units. Viral supernatants are collected twice after transfection, filtered through 0.45 um member, and either frozen down (-80oC) in aliquots, or concentrated to smaller volume, depending on the Env proteins and investigator's preference. A small aliquot is used to titer the virus either by FACS analysis on infected cells (for those expressing GFP) or by Southern blot analysis.
3. Adenoviral vectors:
A. the vectors: The adenoviral vectors provided by the Core are standard E1- and E3-deleted adenoviral vectors. Cloning plasmids are available to express the gene(s) of interest as the single gene product from the vector, or to express them simultaneously with GFP from a bi-directional promoter, which provides a convenient way of monitoring infected cells. A tetracycline inducible vector system is also available to allow for regulated gene expression.
B. virus production: Virus was derived as plaque forming units after transfection of linearized vector plasmids into 293A cells, amplified from a single plaque by serial infection onto 20 15cm plates of 293A cells, and purified by two cycles of CsCl gradient. The final products were titered and the identity of the virus was verified by restriction digest and sequence analysis of the viral DNA isolated from each preparation. The yield is usually 2-4 ml purified virus at the titer of low to mid 10^10 pfu/ml.
4. Adeno-associated virus (AAV) vectors:
A. the vectors: Cloning plasmids are available to express the gene(s) of interest as the single gene product from the vector, or to insert them upstream of IRES-GFP, which provides a convenient way of monitoring infected cells. The gene products can be expressed from a number of internal promoters including CMV, PGK, Ubc, MCK, Desmin and a synthetic muscle specific promoters.
B. virus production: Viruses are generated by three plasmid cotransfection (an AAV rep/cap expression plasmid, an adenovirus miniplasmid containing the required helper function and the vector plasmid) into 293A cells by transient transfection. The method allows for great flexibility in that vectors can be made with a variety of different AAV capsids, including those derived from AAV serotypes 1, 2, 5, and 6, for different host ranges and transduction efficiency.
Virus production is performed in units of "standard preps". A standard prep of retrovirus is defined as the amount of virus generated from 10 15cm plates of transfected cells. Virus particles are isolated from the transfected cell lysates either by Heparin column chromatography (for AAV serotype 2 virus) or iodixanol density gradients (for all other serotypes). The final product is dialyzed against PBS and titered by dot blot hybridization. The yields from such a scale of production are usually several mls of virus at the titer of 10^11-10^12 DNase resistant particles or genome copies per ml. There is not enough adenovirus genome present in the system to generate infectious adenovirus.
Services usually begin with a free consultation session with Dr. Jeng-Shin Lee (jlee@hihg.med.harvard.edu) to discuss the vector options and design. The core also provides plasmid construction services at separate charge (see http://hgti.med.harvard.edu/Chargeback.php3 for the complete price list), but investigators interested in performing plasmid construction on their own can obtain the cloning plasmids and related information with a Material Transfer Agreement at no cost. For more information, visit the HGTI website at http://hgti.med.harvard.edu/.