Screen genome-wide for genes regulating cellular responses or signaling pathways
Identify potential drug targets or diagnostic markers that inhibit, enhance, or activate a selectable phenotype
Find crucial regulatory genes using routine growth selections or flow sorting
High-throughput identification of functional siRNAs by Affymetrix GeneChip® hybridization
Cost-effective genome-wide functional analysis at prices affordable to the average researcher
Choose from shRNA libraries in FIV or HIV-based lentivectors
Also available in plasmid form, you can now produce large quantities of packaged shRNA library in your own lab
SBI’s GeneNet shRNA Libraries encode a pooled set of shRNAs designed against all target genes, and are constructed using a lentiviral delivery system so the library may be introduced into almost any cell type. Upon infection with an shRNA library, a functional screen is applied, and those cells exhibiting the desired phenotypic changes are isolated. The shRNA constructs, presumably inducing the phenotypes of interest, are recovered by PCR and identified by microarray hybridization or sequence analysis. SBI’s GeneNet libraries allow for comprehensive, cost-effective genome-wide RNAi screens to be performed by any research group.
GeneNet™ shRNA Libraries are used to screen for sets of genes functionally involved in a specific biological process or cell response. For example, we have used a GeneNet™ shRNA Library targeted to 1,500 cancer-related genes to identify genes involved in the response to radiation.
Functional gene screening with a ready-to-transduce packaged GeneNet Library involves the following steps that are illustrated above.
Transduction Target cells are transduced with the packaged GeneNet Library. With the high-efficiency, pantropic VSV-G pseudotyped HIV or FIV-based lentiviral system, you can achieve transduced cell populations where every cell expresses at least one shRNA.
Treatment The transduced target cells are then treated to induce the phenotype of interest. For example, the cells may be exposed to radiation, induced to differentiate, enter apoptosis or could be used in a drug screen.
Selection After treatment, cells with the appropriate phenotype are selected using either a growth selection, flow cytometry for specific markers, or other approaches where the population of cells with the desired phenotype can be isolated and collected.
Amplification After selecting the target phenotype cells, the shRNA templates from the library are recovered by PCR. Since the flanking sequences from the library vector are known for all the inserts, these sequences are used to amplify the shRNA templates from either RNA or DNA isolated from the target phenotype population. The shRNA templates from a population of transduced, but untreated cells, will be used as comparison to identify effector shRNAs that are either enriched for or selected out in the screen.
Identification Determining the specific shRNAs isolated from the library screen can be easily done by hybridization to a Affymetrix GeneChip® Arrays that recognizes all the shRNA templates present in the library. To identify critical genes involved in the pathway or response, the shRNAs in the target phenotype population are compared with shRNAs recovered from a control (untreated) cell population. The shRNAs that are over-represented in the phenotype population target genes that regulate the phenotype from the screen. For example, when selecting for radiation resistance, those cells resistant to radiation and surviving the selection presumably contain shRNAs that target genes that would normally make the cells sensitive to radiation. It is these shRNAs that are over-represented in the treated population and can be directly correlated to their targeted genes through sequence identity.
Data analysis support SBI's experienced scientific staff are available for guidance in developing GeneNet shRNA screens as well as for post-screen consultation and data interpretation.
The GeneNet Advantage
SBI’s genome‐wide shRNA libraries have been popular with researchers world‐wide for over 3 years.
All library technology was developed in‐house, and our team of scientists offers unparalleled support and expertise to assist with experimental screen design and optimization.
An shRNA library pool is only as good as the viral delivery system it uses, and SBI’s leading lentiviral technology is world‐class. Our viruses infect better, period. In a side‐by‐side comparison of 3 similar lentiviral preparations from leading private companies, researchers at Stanford University found that SBI’s lentiviruses offered the highest infection efficiency of hematopoietic stem cells [reference available]. Try our positive transduction control virus and see the SBI advantage for yourself.
GeneNet libraries are available as prepackaged virus, so you can begin infecting cells the day you receive the library. Choose from FIV or HIV‐based vectors depending upon transfection efficiency in your cell type.
GeneNet libraries are compatible with standard Affymetrix chips to allow for easy identification of effector sequences. SBI provides a comprehensive data analysis tutorial with all libraries and offers our experienced technical support to assist with interpreting results of screens using our RNAi libraries.
Sample Library Data
A. Screening for Genes Involved in Fas-induced apoptosis
This figure demonstrates the use of SBI's genome-wide shRNA library for the search for genes involved in Fas-induced apoptosis. Red circles represent siRNAs that are enriched only in the Fas-treated samples, and therefore may silence gene(s) associated with the biological pathway(s) in vivo that are associated with apoptosis. Blue circles are siRNAs present in both Fas-treated and control samples.
B. Screening for Genes Associated with Differential Response to a Drug
This figure demonstrates the use of SBI’s genome-wide library to identify genes associated with drug response.
The “Eliminated siRNAs” correspond to those genes that conferred a selective disadvantage in presence of the drug and hence are only present in the Control population.
The “Enriched siRNAs” correspond to those genes that confer a selective advantage in presence of the drug (perhaps enabling cell survival) and hence are only present in the Drug Treated population.
Cells were transduced with a GeneNet™ Expression Library expressing approximately 7,000 siRNA template sequences targeted to 1500 cancer-related genes. Following transduction, treated cells were exposed to high doses of gamma-radiation twice during several days of growth. The control cells were cultured normally. After harvesting and isolating DNA, the siRNA sequences present in both cell populations were amplified and hybridized to the Affymetrix GeneChip® Human Genome Focus Array. A comparison of the hybridization results for the two cell populations is shown.
Each point on the graph represents the signal from one probe on the array after hybridization to each sample. Points on the upper left, above the diagonal represent siRNA sequences that are enriched in the radiation-treated control population as compared to the untreated control cells. The light blue points do not have significant signal on the arrays, and the yellow points do not show a significant enrichment in the treated population. The points in dark blue are siRNA sequences over-represented by 3-fold in the treated population, and the points in red are sequences enriched more than 5-fold in the treated cells.
The siRNA sequences enriched in the irradiated cell population conferred resistance to radiation treatment, so these siRNA sequences target genes that make the cells more sensitive to radiation treatment. Note that these genes are not “up- or down-regulated” as would be the case with standard expression analysis. This assay indicates that the function of these genes sensitizes the cells to radiation and that their elimination improves viability of the cells. Supporting this, three of the 9 genes that are enriched more than 5-fold in the treated population are related to cell growth and control: Meiotic Recombination (MRE11), Tumor Suppressor (DOC1), and Apoptosis (AK3). The other 6 genes that are enriched more than 5-fold are currently being analyzed.
