Identification of modulators of chemotherapeutic resistance using a random shRNA library

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Copyright: Ramadas, Radhika
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Abstract
High-throughput gene silencing using RNA interference (RNAi) libraries is a rapidly expanding strategy to study the molecular pathways underlying human diseases. A majority of RNAi libraries contain expression constructs designed to target specific genes or gene sub-classes. The use of random RNAi libraries offers a more wide-reaching alternative to identify novel therapeutic targets. However, most of the previously reported protocols for the generation of random RNAi libraries remain highly complex. In this thesis, the development of a simplified and efficient method for the construction of a random shRNA-encoding library is reported. To prove the functionality of the random shRNA-encoding library, it was used to investigate the molecular components and mechanisms used by cancer cells to acquire resistance to three anti-cancer drugs from two drug classes with distinct mechanisms of action. Using independent selection assays, cells transduced with the random shRNA-encoding library were exposed to cisplatin, carboplatin or paclitaxel, and resistant cells isolated. A total of 159 unique shRNA-encoding inserts were recovered, sequenced and used to perform homology-based searching of protein- and RNA-encoding databases. Several of the inserts were enriched in cells displaying resistance to the specific chemotherapeutic agents. The predicted target sequences included both previously reported targets implicated in cellular resistance mechanisms and novel targets, thus proving the feasibility of this approach in identifying new targets for therapeutic agents. The cell-based RNAi screen and subsequent bioinformatic analysis produced a list of novel gene targets for further investigation of the role each plays in chemotherapeutic drug resistance.
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Author(s)
Ramadas, Radhika
Supervisor(s)
Arndt, Greg
Dawes, Ian
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Publication Year
2012
Resource Type
Thesis
Degree Type
PhD Doctorate
UNSW Faculty
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