Despina C. Siolas

3rd Year Medical Student

Department: SOM

Graduate Program: Genetics

Advisor: Dr. Gregory Hannon

Abstract:

Title:  High Throughput RNA interference barcode screens as a tool for discovering gene function

Recent advances in the field of RNA interference (RNAi) have enabled researchers to conduct in depth investigations of gene function. High throughput screens in cultured mammalian cells can now be performed using RNAi libraries such as our own Hannon-Elledge library. Our second-generation libraries consist of over 200,000 short hairpin RNA constructs targeting over 45,000 human and mouse genes modeled after primary miRNA transcripts. Each hairpin is linked to a unique 60 nucleotide identification sequence, which serves as a barcode and allows us to virtually count the number of cells that contain a specific hairpin in a cell population. Small changes in barcode copy number can be monitored through the use of microarray technology. The barcode can be amplified from a cell’s genomic DNA and fluorescently labeled to produce a probe that is hybridized to a microarray. We have optimized our probe labeling methods, probe size and hybridization conditions using library plasmid DNA in a Nimblegen platform. This optimized protocol allows us to detect 78.6% of probes within 1 standard deviation above the mean background from a complex mixture of approximately 1500 hairpins. In addition by using two color hybridization (Cy3 and Cy5) we can detect control subsets of hairpins known to be depleted from a sample population. We applied this RNAi barcode screening method in an in vivo screen using a complex mixture of 7500 library hairpins in HCT 116 colon cancer cells to identify genes that modify sensitivity to a common chemotherapeutic, paclitaxel. Hairpin infected cells were treated with paclitaxel at an ineffectual dose (IC20) where 80% of the cells survive. This low dose exposes genes that will increase the sensitivity of HCT 116 cells to the drug by causing an increase in cell death. Genes that synergize with a suboptimal drug treatment should make the drug more potent at a lower dose. Cells that have increased susceptibility to paclitaxel are reflected as a loss of barcode representation on the microarray as compared to DMSO treated control populations. This screen has resulted in a number of viable candidate genes which are currently being validated.

Publications:
(MSTP-supported publications indicated with an *)

*Silva, J.M., Li, M.Z., Chang, K., Ge, W., Golding, M.C., Rickles, R.J., Siolas, D., Hu, G., Paddison, P.J., Schlabach, M.R., Sheth, N., Bradshaw, J., Burchard, J., Kulkarni, A., Cavet, G., Sachdanandam, R., McCombie, W.R., Cleary, M.A., Elledge, S.J., and Hannon, G.J. (2005). Second-generation shRNA libraries covering the mouse and human genomes. Nat. Genet. 37:1281-8.

*Siolas, D., Lerner, C., Burchard, J., Ge, W., Linsley, P.S., Paddison, P.J., Hannon, G.J., and Cleary (2005). M.A. Synthetic shRNAs as potent RNAi triggers. Nat. Biotechnol. 23:227-31.

News:

This paper was announced to be a "ISI fast breaking paper". Please see link for more details.