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Masters Degrees (Genetics)

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Browsing Masters Degrees (Genetics) by Author "Daniels, Aliscia Nicole."

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    Elucidation of gene function using RNA interference in a cancer cell culture model.
    (2011) Daniels, Aliscia Nicole.; Singh, Moganavelli.; Ariatti, Mario.
    RNA interference (RNAi), mediated by small interfering RNA (siRNA), has emerged as a powerful tool for elucidating gene function and also holds great potential for the treatment of acquired and inherited diseases. The delivery of siRNAs still remains a major obstacle for their therapeutic use. Cationic liposomes, a group of positively charged nanovesicles, represent a class of non-viral vectors that have shown the ability to efficiently bind and deliver siRNA. In this study, six cationic liposome formulations containing either cationic cholesterol derivative [N-(N’,N’-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) or N,Ndimethylaminopropylaminylsuccinylcholesterylformyl- hydrazide (MSO9) were prepared with the neutral lipid dioleoylphosphatidylethanolamine (DOPE). Varying amounts of distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), (0, 2 and 5 mole percent) were also included in the liposomal formulations as polyethylene glycol is known to improve the lipoplex bioavailability in vivo. We present evidence that siRNA may be delivered to mammalian cells, in vitro, using a novel cationic liposome carrier system and that siRNA binding and transfection efficiency of the cationic liposomes are affected by the degree of pegylation. Cryoelectron microscopy revealed that the liposome vesicles were unilamellar and were in the 30 - 130 nm size range, while band shift assays confirmed the formation of complexes between the siRNA and the liposome preparations. These siRNA lipoplexes were shown to afford protection to their siRNA cargoes against serum nuclease degradation and were also shown to be relatively non-toxic to the HeLa tat luc cell line which stably expresses the firefly luciferase gene. Cryoelectron microscopy revealed that an inverse relationship exists between the lipoplex size and the degree of pegylation. To determine the transfection efficiency of the cationic liposome preparations in the HeLa tat luc cell line, complexes were prepared with anti-luciferase siRNA, which is specific for the firefly luciferase gene, and knockdown of the luciferase gene was monitored in transfected cells. The results show that liposomes containing the cytofectin Chol-T were particularly effective, achieving up to 93.4% gene knockdown at the 30 nM siRNA concentration. The non- pegylated and pegylated cationic liposomes that have been formulated and examined in this study therefore warrant further development to facilitate in vivo studies.