Methodological Study Of The Delivery Systems Of Small Interfering RNA

RNA interference (RNAi) is a posttranscriptional silencing mechanism triggered by double-stranded RNA. In mammalian cells, small interfering RNA(siRNA)that is shorter than 30bp can trigger sequence-specific degradation of the mRNA to which the siRNA is targeted and not activate ds-RNA-dependent protein kinases. This method, exploiting RNAi, is now widely used to analyze the function of individual genes, screen drugs target, development therapeutic anticancer agents and antiviral agents as a knockdown tool. Currently, the methods to made the knockdown to work can be divided into two category: ⑴ Achieved siRNA by some methods in vitro such as synthetic duplex RNA, then delivery them into mammalian cell to made it to work. ⑵ Another strategy is to delivery recombinant DNA constructs which can express similar short interfering RNA duplexes within cells. However, whatever strategy you choose, a efficient delivery system is indispensable, which is efficient, non-toxic gene carriers that can encapsulate and deliver foreign genetic materials into specific cell types In this study, we aimed at exploiting efficient delivery system to introduce siRNA into cultured mammilla cells. Haven kept those advantages and disadvantages in mind, we carried out experience in both biological gene delivery systems (viral vectors) and Non-biological gene delivery systems. With regard to Non-biological gene delivery systems, we funded that Cationic liposome Dc-Chol/DOPE had potential to delivery short duplex DNA or RNA into cells through endocytosis pathways and to release encapsulated DNA or RNA into the cytoplasm from endosome. It will be very useful for RNAi. With regard to viral vectors, this study developed a new method to produce recombination adenoviral particles by transfect HEK293 cell line with Cationic lipids Dc-Chol/DOPE, which is different and more efficient from classic Ca3(PO4)2 method. Moreover, in this study we also research on the protein transduction peptide, as well as it's effect in gene delivery of recombinant adenovirus. And we fund the improvement of cellular adenovirus uptake with TAT peptide is cell-type dependent. This finding offers not only a evidence that protein transduction may likely depend on receptor molecular on the surface of cell membranes, but also the promise of using the technology to transfect specific cell types.