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.