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RNAi Rescue Screens

Cellecta RNAi rescue screen flowchart

Cellecta RNAi rescue screen flowchart

Identify Genes Functionally Required for Cell Sensitivity to a Treatment or Compound

  • Find genes required for cell sensitivity to a specific treatment or compound
  • Identify pathway or gene clusters mediating a specific cell response
  • Elucidate the mechanism of action or target pathway for a compound or drug

RNAi screening with Cellecta complex pooled shRNA expression libraries requires a strong cell-based phenotypic selection where a distinct population can be isolated from a population of affected cells. The most straightforward approach for this sort of selection is a treatment, such as a drug, that kills almost all cells. If this treatment is applied to a population infected with one of our heterogeneous lentiviral shRNA expression libraries, it is possible to identify the genes that are functionally required for the treatment to generate the effect. This type of screen can be very useful for identifying the biological mechanism of action (MOA) or targets required for a lead compound or other small molecule.

How Does a Rescue Screen Work?

Conceptually, the process is straightforward: infect cells with the shRNA expression library, treat the cells with a factor that under normal conditions kills virtually 100% of cells, then determine which shRNA are present in the surviving cells. Presumably, the genes targeted by the shRNA in the surviving cell population are essential for eliciting the killing response caused by the compound, factor, or small molecule since silencing the gene prevents the cell-death signal from propagating. Thus, analysis of the “rescued” survivors indicates which genes are necessary for the lethal activity of the compound or factor.

An example shown in the TGF-β Screen Application Note (PDF) uses TGF-β to to induce apoptosis in cells. A rescue screen is performed to identify genes required to produce the apoptotic response with this factor. Cells are infected with a lentiviral library expressing approximately 27,000 shRNA targeting approximately 5,000 genes, then the cells are subsequently treated with the apoptosis-inducing TGF-β. After a period of incubation during which most of the cells apoptose, the shRNA in the surviving population is compared with the shRNA in the original library to identify which ones confer resistance to the treatment. When done under appropriate conditions with sufficient replicates, shRNA appearing in the the surviving cells confer apoptotic resistance presumably by interfering with expression of its target. The target genes, then, are functionally necessary for the apoptotic response.

Synthetic Sensitivity RNAi Screens Are Often Combined with Results from a Rescue Screen

Results from this sort of rescue screen can be combined with results from a synthetic sensitivity RNAi screen—a variation of a viability screen where cells are subjected to non-lethal treatment (e.g., a low dose of a lethal compound). With a sensitivity screen, shRNA can be identified that make cells more susceptible to a treatment, thereby indicating which genes help cells survive in the presence of the factor or compound used in the treatment (generally lethal genes are screened out by differential analysis of shRNA that kill cells not subjected to any lethal treatment—a standard viability screen). Following this screen, genes that make cells more sensitive to a treatment can be compared with the genes required for treatment to function in order to generate a more comprehensive view of the genetic pathways associated with the mechanism of action for the lethal drug or factor. FAS induced apoptosis RNAi rescue screen scatter plot

Let Cellecta’s Expert Scientists Help You!

Of course, few treatments are 100% lethal, particularly since it is usually optimal to run the assay right at the borderline where the treatment is highly lethal but survivable with small adjustments to cellular biology. Under these conditions, there are always some cells in a large population that may be resistant to the treatment—even in clonal populations—and others may acquire resistance though spontaneous mutation. So, although the assay is conceptually straightforward, an RNAi rescue screen requires careful attention to the experimental design, including ensuring that sufficient replicates are run, using sufficient cells and virus to get appropriate numbers of infected cells, and optimizing the treatment and cell growth conditions.

The price for this type of screen typically depends on the treatment variations, number of cell lines, types of cells, and related specifics. If you are interested in having Cellecta run an RNAi screen to identify genes critical for the response of interest in your biological system of interest, please contact us