| RNA interference (RNAi), also called post-transcriptional gene silencing (PTGS), is a process in which double-stranded RNA (dsRNA) induces the post-transcriptional degradation of homologous transcripts in sequence-specific manner. It is believed to have evolved as a conservative host defense mechanism directed at transposable elements and infecting viruses. In mammalian systems, introduction of 21-23 nt RNA duplexes as small interfering RNA (siRNA) into host cells leads to high-efficient and specific depression of endogenous or exogenous target genes, but without triggering non-specific responses by interferon pathway. This discovery significantly advances the field and develops RNAi technology as a powerful tool for the studies of protein functions, anti-viral therapy, and cancer research. However, the effective of siRNA is highly dependant on accessible site, although there have be some rules for selection of siRNA sites can be followed, all the selected candidate siRNA must be tested its RNAi effect and screened out optimal siRNA through time-consuming and laborious experiments. Therefore, it is necessary of establishment of high-efficiency and economic platform for identification siRNA.Firstly, a quick and easy method to obtain siRNAs is warranted for the ideal screening system. Short siRNA molecules can be prepared by chemical synthesis or in vitro transcription. Alternatively, they can be transcribed in vivo by using siRNA or shRNA expression vectors with a RNA polymerase(pol) III promoter (including U6, human HI, and tRNA promoters), or a pol II promoter with a minimal poly(A) signal sequence. In addition to plasmid- and virus-based systems, two-step PCR-derived short hairpin RNA (shRNA) expression cassettes (SECs) have been shown to efficiently suppress transfected gene activity and proved to be a rapid, facile and cheap approach foridentification optimal siRNA. However, PCR mutations in the siRNA sequence will be increased bythe two-step PCR approach and then, its high efficiency of RNAi would be impaired. In Part I of this report, we attempted to develop a single-step overlapping exunsion PCR for yielding high quality SECs. Using enhanced green fluorescence protein (EGFP) as target to test RNAi effect of the PCR-based SEC strategy.An easy and standard RNAi effect reporting system is also required for a large scale screening tasks. Fluorescence reporting systems have been used for analysis of RNAi effect, by which the target gene can be expressed with fused fluorescence protein and easy to be detected. In Part II of this study, we constructed recombinant expression plasmid fused with EGFP and MBx, and established a system for screening of HBx-siRNA with fluorescence protein-based reporting systems. The SECs with efficient siRNA and matching promoters screened out from this system were then transfected into a stable HBV-producing cell line HepG2 2.2.15 cells to evaluate its inhibition effect on HBV gene expression in vitro.Mouse La protein (mLa) has been identified as a host factor for stabilizing HBV RNA. Recently, the human La protein (hLa) was characterized to bind HBV RNA with high affinity implied that hLa might be involved in HBV RNA metabolism. But the role of human La protein (hLa) in HBV RNA metabolism in vivo is still unknown at present. In Part III, we prepared SECs targeted La protein, an endogenous human cell gene, and transfected them into HepG2 2.2.15 cells to investigate the correlation between depletion of hLa function and inhibition of HBV expression. The therapeutic feasibility of RNAi specific for the human La protein, a putative cellular cofactors for HBV was discussed.Part I Quick generation of PCR-basea shRNA expression cassettesMETHOD AND RESULTSPreparation of SECs by single-step overlapping extension PCRFor generating template used in PCR for preparing SEC, the same flanking sequences were added to each side, of three RNA Pol III promoters (U6+I, HI and tRNAVal) by PCR method. Via overlapping extension single-step PCR, two universal primers and one g...
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