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

peutics, often combining paclitaxel with one, or sometimes two, additional chemotherapies, including cisplatin, carboplatin, or radiation treatment.

Since chemotherapy is toxic and stressful to a patient, there is an obvious benefit to limiting the amounts of these toxic compounds administered. If patients who are likely to positively respond to paclitaxel as a single agent could be identified prior to treatment, they may be spared the unnecessary pain associated with combination regimens. One potential way to do this is through employment of pharmacogenomics.

The first step in a potential strategy would be to categorize patients based on the molecular profile of their tumor. This can be effectively accomplished through use of microarrays (on an RNA level), array CGH (on a DNA level), or protein chips. This type of work is well established and has been thoroughly reviewed elsewhere.

After understanding the profile of a tumor, one still must determine which molecular signature is indicative of responsiveness to a drug, and which is not. We utilized an shRNA screen to help elucidate this question, specifically in the case of NSCLC and paclitaxel treatment.

Various transcripts were down-regulated using lentiviral-based shRNAs found in a panel targeting tumor suppressor genes (Sigma-Aldrich, MISSION ↓ TRC shRNA Human Tumor Suppressors, SH0531) in lung cancer cells grown under standard conditions. Transductions were performed in 96-well plates, with each well receiving cells and a single shRNA delivered by lentiviral particles. The entire tumor suppressor panel, consisting of approximately 75 gene targets each represented by 3-5 individual shRNA clones, fits onto five 96-well plates. After selection of transduced cells with puromycin, each well was split1:2 (2 sets of 5 x 96-well plates). One set of plates was mock-treated while the second set was treated with paclitaxe
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