shRNA Resource

Introduction:

The Molecular Screening  Facility maintains genome scale resources for RNA interference directed regulation of gene expression.  The facility also provides technical support to Wistar Scientists engaged in somatic cell “Loss of Function” experiments for target identification and mechanistic studies to dissect the role of genes, signaling pathways in human disease pathologies.

Libraries:

The Wistar Institute has purchased bacterial glycerol stocks of the complete (human and mouse) TRC shRNA library (Moffat, J.  et al Cell, (2006) 124:1283-1298). The human library (TRC-Hs1.0) targets 15,000 annotated human genes and consists of 80,700 precloned constructs.  The mouse library (TRC-Mm1.0) targets 15,000 annotated mouse genes and consists of 76,800 precloned constructs.  The hairpin sequences, a 21-base stem and a 6-base loop, are each cloned into the pLKO.1 vector, which allows production of replication-incompetent lentiviral particles, transient or stable expression of the shRNA, and antibiotic (puromycin) selection of transfected or infected cells.  On average, each gene is targeted by ~5 constructs.  The shRNA sequences for a given target are distributed throughout the cDNA sequence of a target, including a shRNA clone targeting the 3'UTR for use in phenotypic rescue studies using cDNA expression constructs.  For more information about the library, including shRNA target selection and genes targeted, vist http://www.broad.mit.edu/rnai/trc/lib.

Services

The Molecular Screening Resource provides three services related to distribution of clones from the TRC shRNA library.

Individual clone distribution: Cancer Center members can request shRNA clones from the TRC library.  Clones can be ordered through a web-based system accessible through the Wistar Intranet. For ordering instructions, click here.

The Protein Expression facility will prepare custom, VSVg pseudotyped vectors.  For more information, click here.

Whole genome pooled library: The Resource has prepared pools of plasmid DNA for clones from the TRC shRNA library by species and produced concentrated viral vector (³5x108 TU/ml) for infection of mammalian cell cultures. Each plasmid DNA/viral vector pool consists of ~9600 constructs with the whole genome of each species (human and mouse) covered by 12 pools, respectively. The pools are not organized by GO annotated gene families. Each viral shRNA vector pool and control vector (e.g. empty, non-targeting shRNA) will be provided as 2 x 10ul frozen aliquots in PBS that have been verified to be at least 5 x 108 TU/ml, as determined using a p24 assay.

Focused shRNA Gene Sets: The Resource will format clones of focused gene sets (i.e. user defined, GO annotated gene family, etc.) in new 96 well microtiter plates for growth and preparation of plasmid DNA. 

Control Clones

The Molecular Screening Facility has assembled a number of shRNA control clones for use in shRNA experiments. For clone information, click here.

Guidelines for controls

Basic Controls:  At a minimum, the levels of the target mRNA and/or protein, when possible, should be assessed using appropriate quantitative methods (e.g. qRT-PCR, western blotting, etc.)

Multiplicity controls: One way to enhance the overall confidence in RNAi data is to demonstrate a similar effect with two or more target sequences that localize to different regions of the mRNA under study.

Functional controls:  The ultimate control for any RNAi experiment is to rescue a resultant phenotype of a sopecific siRNA by physiological expression of the target-mRNA in a form that is refractory to the siRNA.  This approach can often be realized by utilizing one or more silent point mutations in the third codon position within the targeted region of the native mRNA, utilization of a siRNA targeting the 3’ UTR of the message, or expression of a functionally equivalent cross species mRNA. 

Policies:

1. Principal Investigator requesting clones must have a signed copy of the Limited-Use license on file in the Institutions Office of Business Development.

2. Clones and/or virus may not be distributed to scientists outside of the Wistar Institute.

3.Wistar materials, defined as cell lines or model organisms derived from use of the TRC shRNA clone may be distributed in accordance with the requirements of the Institute’s Materials Transfer Agreement.

4. The Molecular Screening Facility does not guarantee the authenticity of the clones in a given library plate/position or the effectiveness of clones to knockdown the expression of a specific target gene.

5. It is the responsibility of the investigator to register the intended specific use of recombinant DNA technology (i.e. retrovirus use) with Wistar’s Institutional Biosafety Committee (IBC) as required by the most current NIH Guidelines for Research Involving Recombinant DNA Molecules.  For a copy of this document, click here.  For a full description of requirements, please consult the NIH Guidelines website.

6. Data published from the use of specific clones should indicate the TRC clone # (TRC000000xxxx) in the manuscript.

7. It is expected that Investigators will acknowledge the Molecular Screening Facility in manuscripts, when appropriate.