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A Screen of shRNAs Targeting Tumor Suppressor Genes to Identify Factors Involved in Paclitaxel Sensitivity

Diana Ji, Stacy L. Deeds, and Edward J. Weinstein


RNA interference methodologies have been utilized in small to large scale screening projects. The technology has allowed researchers to perform gene-silencing experiments in timeframes and target cells previously not possible. While siRNA screens have become fairly common, large scale screening with shRNA is still evolving. Advantages of shRNA-based experiments include long-term knockdown and viral delivery to non-transfectable cell types.

We set out to develop strategies for using lentiviral-based shRNA libraries in larger scale silencing projects (see Figure 1 for library vector map). Pilot screens using a tumor suppressor gene family set were performed to address biologically relevant questions while simultaneously developing screening strategies that could be used by a variety of researchers in the field.

A topic that has been extensively investigated is the effectiveness of chemotherapy for treating various carcinomas. A major cause of mortality in the United States, lung cancer, accounts for approximately one third of cancer-related deaths and has a low 5-year survival rate with few patients responding effectively to chemotherapy.

One potential treatment for lung carcinoma is paclitaxel. This anti-cancer agent (produced by Bristol-Meyers Oncology) functions by stabilization of microtubules so they cannot depolymerize. It effectively shifts the equilibrium in cells towards microtubule assembly, disrupting the normal operation of the microtubule network and thereby arresting the mitotic process.

Treatment with paclitaxel alone, however, is effective in only a fraction of the population. Approximately 21% to 24% of patients with non-small cell lung carcinoma (NSCLC) will respond to a regimen with this single agent. As a result, first line therapy may involve multiple chemothera
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