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shRNA Core Facility
Irwin Gelman, PhD, Director
Contact info:
Consultation*:
| Renae Holtz Irwin Gelman, PhD |
* Please contact for questions regarding experimental design.
Description
Sequence validated human and mouse shRNA libraries in virus vectors are available through the shRNA Core Facility. The shRNA library permits rapid, cost-efficient, loss of-function genetic screens and rapid tests for genetic interactions to be performed in mammalian cells. Each shRNA construct has been sequence verified and cloned into a retroviral vector to ensure a match to the target gene. Each gene is represented by a target set that consists of an average of 3-5 individual constructs or clones targeting different regions of the gene sequence.
In addition we have acquired the Human Cancer Retroviral Library. The Expression Arrest™ Human Cancer shRNAmir Retroviral Library targets all cancer related genes, including known tumor suppressor genes with multiple shRNAmir constructs per gene. The library contains over 2311 shRNAmir constructs targeting 967 cancer related genes.
The shRNA expression cassette is carried in a validated Murine Stem Cell Virus (MSCV) backbone. The pSHAG-MAGIC 2.0 vector can be used both for transient and stable delivery by transfection using the replication deficient retrovirus as a delivery method.
Additionally, the libraries have an incorporated DNA “bar-coding” system, by which each RNA molecule has been tagged with a unique 60 nucleotide DNA sequence. This facilitates shRNA validation and gene identification after library screens.
The shRNA libraries were purchased from Open Biosystems. The microRNA-adapted shRNA (shRNAmir) constructs are modeled after primary microRNA transcripts allowing efficient processing via the endogenous microRNA pathway and producing increased, more specific knockdown. Designed with features aimed at increasing gene knockdown efficiencies and versatility of use in RNAi screens, shRNA libraries provide solutions for diverse RNAi applications.
Please visit the following sites for more information on the Expression ArrestTM shRNA, Human, Mouse, and Human Cancer Gene library collections. In addition, please refer to the recent Nature Genetics Technical Report for information on the libraries.
Location
Buffalo Life Sciences Complex (BLSC) Room L2-135
Operating Schedule
Weekdays, 8:30 am - 4:30 pm
Using the Facility
- To place an order, you will need the official gene name. This is available at the NCBI website.
- In the search window select “gene” and type gene name or alias.
- Once you have the official name, you can go to the Open Biosystems website to search for the Oligo ID number for your clone(s).
- Type the offical gene name in the search window and submit. Click on shRNAs. Select for the shRNAmir clones in the pSM2c retroviral library format.
- Click on the oligo ID number of each selected clone for the "Cloning Details."
- Please include the Clone ID# and Location when e-mailing us with your request(s).
- Requests for clones and questions about pricing and availability can be sent to renae.holtz@roswellpark.org.
Specific services
Upon receipt of an order, bacterial cultures will be amplified from the glycerol stocks for use in purification of plasmid DNA. Retroviral particles will be produced from the plasmid DNA by transfection of either ecotropic, amphotropic, or polytropic packaging cells. The shRNA Core will then help the researcher with the infection of target cells. The Core can also select for individual or pooled puromycin resistant colonies, and after expansion, these cells will be provided back to the researcher.
The reagents could be used to study the effects of knockdown of individual genes or groups of genes in cultured cells, in vivo, or in transgenic mice after infection of ES cells. The genomic shRNA retrovirus libraries can be used to screen for genes involved in various biological functions.
Our Core can also produce ecotropic, amphotropic, or polytropic retroviral supernatants from DNA constructs provided by researchers. A similar service is provided for lentivirus production. Costs for other transfection services and RNAi resources will be determined in consultation with shRNA Core personnel.
Billing Procedure
The investigator’s grant is charged for services.
RPCI / UB User fees
Glycerol stock: please contact*#
Plasmid DNA: please contact*#
Retrovirus production for infection of cell line:
- each clone: $75.00
- 10-25 clones: $60.00/clone
- 100-500 mixed clones (e.g. all kinases:) $35/clone
- mixed clones for whole genome: please contact*
Infection of cell line and puromycin selection:
- each clone: $150.00
- 100-500 mixed clones (e.g. all kinases:) $1000
- mixed clones for whole genome: please contact*
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* Please contact Renae Holtz for information.
# Individual labs who wish to have unlimited access to the libraries can pay a one- time licensing fee of $1000.
● Non RPCI/ non UB users who wish to use the facility please contact Renae Holtz for more information.
