Human shRNA Library Screening to Dissect Pathways Involved In Telomerase Actions

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Title: Human shRNA Library Screening to Dissect Pathways Involved In Telomerase Actions
Author: Hoshiyama, Hirotoshi
Abstract: The minimal components of human telomerase are the human telomerase reverse transcriptase (hTERT ) and the human telomerase template RNA (hTR ) . Although it is known that both components are minimally sufficient to reconstitute telomerase activity , the factors involved in any of the multiple steps of telomerase action such as telomerase assembly , telomerase recruitment to telomeres , and telomere extension /regulation are not well understood . There are a large numbers of proteins that have associations with telomerase , yet the functional roles of those in telomere maintenance and telomerase regulation are not well understood . Identifying novel proteins and pathways involved in any of these important telomerase -associated functions will be useful for identifying new targets for the development of novel inhibitors that block telomerase function in cancer cells . Therefore , my goal has been to develop methods to dissect these molecular pathways and identify functional factors involved in any step of telomerase actions . To accomplish this , I designed a selective screening system by exploiting lentiviral shRNA libraries and tetracycline inducible -hTERT cell lines that is hTR deficient but expressing mutant hTR . Thus , the overall strategy of the screening system may be considered a “synthetic rescue screen” . In brief this screen was set up to rescue cells from apoptotic death due to mutant sequence incorporation at telomeres by reducing the gene expressions with lentiviral shRNA libraries . This allows us to look a set of genes involved in pathways involved in functional aspects of telomerase actions , not based on structural association with telomerase . During the work , I have established multiple lines of inducible -hTERT cells to use in selective screening systems . I have also developed a method for rapid construction of high -complexity custom shRNA libraries for targeted screening and re -evaluate hundreds of primary candidate genes to identify smaller numbers of secondary candidate genes by removing false positives . In order to analyze the pooled shRNA screening result , I have developed a method for quantitative identification of half -hairpins from a pooled shRNA library based on the pGIPZ vector . Introducing multiplexing codes and refining sample preparation schemes resulted in the predicted ability to detect two -fold enrichments followed by massive parallel sequencing . Development of those methods allowed me to identify several candidate proteins , which may be involved in telomerase actions .
URI: http : / /hdl .handle .net /2152 .5 /944
Date: 2011-12-12

Citation

Human shRNA Library Screening to Dissect Pathways Involved In Telomerase Actions. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /944 .

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