sh-RNA Library


Obtaining shRNA clones (Target Clones) and relevant virus particles (Target Vectors) from the Lenti-shRNA Core Facility:


Lentiviral shRNA vectors for a known human or murine gene:

• The gene accession number (such as NM_005636) of the relevant target gene will be provided to the shRNA core by the investigator.
• The Core will identify the relevant shRNA clones in the shRNA libraries.
• The Core will provide a bacterial glycerol stock containing the relevant shRNA clones.
• If the investigator decides to get vector particles from the shRNA core, he/she will provide the DNAs (at least 50 ug. DNA concentration should be at least 0.5 ug/ul).
• The requested shRNA vector will be packaged into vector particles. The core will generate a total of 10 ml of filtered conditioned media containing vector particles at titer higher than 106 IU/ml. Vector production can be scaled up to 100 ml or more, as requested.
• Unless requested, vector titers will not be determined.

Virus Particle Production

• Three-plasmid calcium phospate-based transfection into 293T cells
• Vector particles are collected from conditioned media and filtered at 36-60 hours post transfection
• Vector particles can be concentrated by ultracentrifugation

 

Lenti Vector Production Image

 

 

 

 

 

 

 

 

 

Virus Particle Titering

• Vector titers (as IU/ml) can be determined by puromycin-selection and colony counting following serial dilutions on 283T cells.
• Vector titers (as physical particles.ml) can be determined by p24gag ELISA

 

Highly concentrated vector:

Upon request, 100 ul of high titer concentrated virus particles (~ 10 9 iu/ml) concentrated virus (in PBS) can be produced. This service includes vector tittering by p24 ELISA and three independent safety assays to rule out the emergence of replication competent retroviruses (RCRs). We recommend it for in vivo studies.  Please contact the core for a quote.

 

 

 

Partially concentrated vector:

We can provide 400ul (8x50 ul) of partially concentrated vectors (titer> 10^7 IU/mL)  . We recommend this service for in vitro studies requiring high vector titers containing no or very low levels of FBS.  Please contact the core for a quote.

 

 

Obtaining lentiviral shRNA particles for a targeted gene:

• The gene accession number of the relevant gene target will be provided to the shRNA core by the investigator.
• The core will identify the relevant shRNA clones in the shRNA libraries.
• Based on the core’s recommendations, the investigator will determine which viral clones will be generated and at what scale.
• The core will amplify and purify the relevant plasmid DNAs. (charged under the Special Construction rate).
• The core will package the requested shRNAs into vector particles. A standard of 10mL, containing non-concentrated vector at a titer of, at minimum, 106 IU/mL, will be generated. Upon request, vector production can be scaled up to 100 mL or more.  The Target Vector rate is charged for the standard 10ml, and the vector scaled up would be charged as a single Vector large scale.

Controls

We offer a control clone, a control vector, and a large scale vector control. The controls include a plasmid encoding a lentiviral vector containing an empty shRNA expression cassette (a U6-promoter without the shRNA sequence) and a plasmid encoding a lentiviral vector containing an shRNA expression cassette directed to the green fluorescence protein coding region. Controls are needed in all shRNA experiments to rule out of target effects. The controls function as a baseline comparison tool.


Custom Construction Service

An hourly construction charge is available for custom cloning.  If the investigator provides their own lenti viral or Retro viral DNAs, we can package it into 10ml viral particles.