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Home Page |GeneNet RNAi Libraries |Application Overview |Sample Data

Application Overview

Pooled lentivirus shRNA libraries

High-throughput RNAi

  • Dissect signaling pathways
  • Discover new drug targets
Overview
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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.