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Study on hTERT Targeting amiRNA Screen and Optimization of Its Expression in scrAAV Vectors

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Tutor: XuRuiAn
School: Huaqiao University
Course: Biochemical Engineering
Keywords: RNA interference,artificial microRNA,artificial microRNA vector,small RNA deep s
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Type: PhD thesis
Year:  2013
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Abstract:
INTRODUCTION:RNA interference (RNAi), a target specific and potent gene regulating approach, has been extensively exploited for gene therapy and other studies. Despite the conventional short hairpin RNA vector (shRNA) has successfully extended the lifespan of small interesting RNA molecular (siRNA) in hostcells, its serious side-effects are undesirable. Therefore, a safer siRNA expression vector (amiRNA) has been introduced based upon the mechanism of endogenous miRNA biogenesis. Unfortunately, the amiRNA vector could not express the carried siRNA as efficiently as we expected. Thus, it is urgently for us to further improve its siRNA expression, and the optimization of amiRNA vectors would boost development of RNAi based gene therapy.OBJECTIVE:The aim of this study is to overcome current obstacles in amiRNA vectors and enhance its expression for clinical application.METHODS:(1) The human telomerase reverse transcriptase (hTERT) gene targeting amiRNA was designed by the "Block-iT RNAi Designer" software;(2) qPCR, Western-Blot, Telochaser, cell migration test, cell apoptosis detection, angiogenesis assay and tumor transplantation in nude mice technologies used to analyze the gene silencing effects and anti-tumor abilities of the interesting amiRNA;(3) The specificity and safety of interesting amiRNA were analyzed by report gene silencing and gene chip technologies;(4) The miRNA candidate spectrum was fixed on by analyzing their read length, sequence logo, free energy and other important features;(5) The optimal amiRNA candidates were figured out through comparing the RPM value of their guide strands;(6) The scrAAV-amiRNA plasmid was constructed by recombining the interesting amiRNA, amiRNA backbone and self-complimentaryadeno-associated virus (scrAAV);(7) The scrAAV-amiRNA virus was produced by triple plasmidscotransfection method;(8) The quality of produced scrAAV-amiRNA virus was confirmed by realtime PCR and PAGE technologies;(9) The expression of carried amiRNA was judged by stem-loop RT qPCRmethod;(10) The expression profile of interesting amiRNA in certainscrAAV-amiRNA virus was built by small RNA deep sequencing approach.RESULTS:(1) Two hTERT targeting amiRNA sequences which with specificanti-cancer activities were screened out;(2) The interesting amiRNA expression from thescrAAV-pcDNA6.2-amiRNA built on the commercial amiRNA backbone wasnot sufficient to silence its target gene;(3) amiRNA backbone from different species could be interchangedaccording to their phylogenetic distance;(4) The interesting amiRNA expression from the optimizedscrAAV-opt-amiRNA vectors was six times higher than that from thescrAAV-pcDNA6.2-amiRNA;(5) scrAAV-opt-amiRNA expressed more iso-amiRNA sequences thanthose of the functional amiRNA sequence.CONCLUSIONS:(1) It was feasible to optimize the amiRNA vector through miRNA deepsequencing profile analysis;(2) The performance of optimized amiRNA vectors should be judged inlight of its functional mature amiRNA sequence.
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