UCL Cancer Institute

Protocols

Retroviral packaging

Please note, that all procedures involving retrovirus (either Gamma or lenti) must be performed in level 2 containment facilities and you should contact your safety officer prior to beginning this work to ensure you have all the appropriate risk assessments and safety precautions in place. The protocols below do not cover all the safety procedures required.

We have two in-house methods for packaging your hairpins into retrovirus. These protocols can be used for both the GIPZ (lenti) and the pRetroSuperCam (Gamma) hairpins. Although please note that the packaging vectors used are different for the two systems and you will see this indicated in the protocols and outlined below.

PEI based method:

Originally adapted from Adrian Thrashers laboratory within the Institute of Child Health.

• Lower titre virus
• Less expensive
• Retrovirus Production-PEI method UPDATED 07/07/2010

Fugene based method:

Originally adapted from Greg Towers laboratory within the Windeyer Institute.

• Higher titre virus
• More expensive
• Retrovirus Production- Fugene method UPDATED 07/07/2010

Packaging vectors:

Both methods are based on the use of Generation 2 compatible retroviral vectors. Two packaging vectors are used for both methods alongside the vector containing the hairpin of interest.

Both library vectors use the same VSV-g envelope expressor plasmid:

pMDG

However, they use different gag-pol expressors:

GIPZ hairpins:
p8.91

pRetroSuperCam hairpins:
MLV-GP

Retroviral Concentration

You may need to concentrate your virus for use. Here is a ultracentrifugation method to concentrate your retrovirus.

Retrovirus concentration NEW 07/07/2010

Retroviral Titering

It is important to titre your virus so you know how many viral particles or transducing units/ ml (TU/ml) you have. This allows you to determine the amount of virus to use to obtain a specific MOI (Multiplicity of Infection) with your cells.

We recommend titering your retrovirus using a biological based method. Please find a method below for titering your virus using fluorescent activated cell sorting (FACS).

Retrovirus titration NEW 07/07/2010