One strand of the miRNA duplex is then loaded into the RNA-induced silencing complex (RISC), which mediates gene suppression through mRNA degradation or translational repression with the miRNA binding to the target mRNA (Ambros, 2001; Bartel, 2004).
siHBsAg-mediated gene suppression is in a dose-dependent manner with 1 nM of siHBsAg1 being sufficient to inhibit HBsAg accumulation in cells.
This strategy has been used in trypanasomes and Drosophila for generating long dsRNA and inducing RNAi-mediated gene suppression [ 13- 15].
RNAi is the mechanism of sequence-specific gene silencing by either the specific degradation of homologous mRNAs or transcription-mediated gene suppression (1- 4).
Previous studies indicated that longer gene structures were most likely linked to gene regulation, such as splicing regulation and chromatin-mediated gene suppression from introns [ 46, 54].
This type of analysis has been performed before and is enabled by the finding that the majority of total miRNA-mediated gene suppression functions through mRNA degradation, thus functional miRNA activity can be determined by measuring the expression of their targets at the transcript level [ 27– 36].
Additional pathways suggested include: endocrine disruption, suppression of hormone regulation and hormone mediated gene transcription, alteration of cell cycle kinetics, and alterations in cellular proliferative response that might play roles in association with carcinogenesis [ 37].
To determine whether the convergent U6/H1 expression cassette could produce functional siRNA that mediated effective gene suppression, an insert encoding a p53-specific siRNA [6] was enzymatically-generated (as described in materials and methods) and cloned into pHybrid (Figure 1A).
In addition, in vitro synthesis of ~21-nt small interfering RNAs to mediate gene-specific suppression in mammalian cells have been developed in order to extend this particular technique to higher eukaryotes [ 8]. 80% of C. elegans genes have human homologs [ 9].
The above results indicate that the use of U6 convergent promoters in a single expression cassette can produce complementary sense and antisense RNAs that mediate specific gene suppression in a manner consistent with RNAi.
