Gene Transfer, Targeting and Therapeutics
Viral Vector Core

Services

• Production of custom lentiviral, retroviral, rAAV, G-deleted rabies, and adenoviral vectors specific for individual investigators needs
• Production and distribution of stock lentiviral, retrovial, rAAV, G-deleted rabies, and adenoviral vectors expressing reporter transgenes
• Consultation on experimental design, development and use of custom viral vectors, and production of client specific viral transfer plasmids
• Production and distribution of rAAV2 serotype kits containing a range of cross-packaged vectors – Useful for pilot studies prior to custom vector production
• Distribution of cloning vectors and relevant MTA information to investigators prior to vector design and production
• Distribution of validated and titrated iPS reprogramming vector kits

What to provide to the core:

1. 200ug (preferably >1ug/ul) of your lentiviral or rAAV plasmid. If large scale rAAV production is requested, 500ug of rAAV plasmid is required. DNA should have been purified using an endotoxin-free protocol (e.g. Endo-free maxi/mega/giga plasmid purification kits or equivalent). We do not accept miniprep purified DNA.
2. Plasmid DNA should be checked for purity and have an A260/280 of >1.8.
3. AAV ITR plasmids should be checked for recombination by digestion with SmaI or XmaI. Each ITR contains two SmaI/XmaI sites – digestion will cut out your insert. Excessive amounts of linearized full-length plasmid indicate recombination has occurred.
4. Lentiviral and retroviral transfer plasmids should be digested with appropriate restriction enzymes to confirm correct insert size.
5. Please provide a gel image of your SmaI or XmaI digested AAV2 ITR plasmid, or your lentiviral transfer plasmid with the insert excised.
6. To avoid recombination we recommend transforming and growing your lentiviral and rAAV transfer plasmids in recombination deficient cells such as STBL3 @ 30°C IN 2XYT broth for no more that 16 hrs with shaking.
7. Vector map and Sequence file – we encourage you to submit your plasmids to our vector library. We will be happy to work with you on establishing MTA agreements for your reagents.
8. Information about your gene of interest or insert and any special requirements for handling e.g. toxicity, oncogenic, pro-apoptotic, etc.

Protocols

Lentiviral Vectors

• Production of third generation VSV.G pseudotyped HIV-1 Lentiviral vectors
• Production of second generation VSV.G pseudotyped HIV-1 Lentiviral vectors
• Modified production of Rabies glycoprotein pseudotyped EIAV lentiviral vectors
• Modified production of HIV-1 lentiviral vectors pseudotyped with Non-VSV glycoproteins

Retroviral Vectors

• Production of VSV.G pseudotyped MMLV (MSCV) retroviral vectors

rAAV Vectors

• Production of recombinant cross-packaged rAAV vectors using density gradient purification
• Secondary purification of rAAV vectors following density gradient separation