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At least 1,081 records · Page 60Linked to original sources
PubMed2005

[Inhibition of HBsAg and HBeAg secretion by RNA interference of the polymerase gene sequence of hepatitis B virus: an experimental study].

OBJECTIVE: To investigate the effects of small interfering RNA (siRNA) targeting the polymerase (P) gene sequence of hepatitis B virus (HBV) on the replication and antigen secretion of HBV. METHODS: From the 29 base sequences of the HBV in the HepG2.2.15 cells that accord with the demands of siRNA designing five sequences targeting the P gene of HBV were selected and cloned into the siRNA expressing vector pGE-1. Then the plasmid pGE-HBVP was transfected into the cultured HepG2.2.15 cells. Chemiluminescent immunoassay was used to determine the levels of HBsAg and HBeAg in the supernatant of culture medium 24, 48, 72, and 96 hours after the transfection and the expression of HBsAg in the 2.2.15 cells 24 hours after the transfection so as to observe the inhibitory effects. Untransfected cells and cells transfected with blank pGE-1 vector were used as controls. RESULTS: Five vectors expressing the siRNAs targeting the HBV P region, pGE-HBVP1-pGE-HBV5 were successfully constructed. The efficiency of transfection of the vectors into the 2.2.15 cells were 30% to 40%. 24, 48, 72, and 96 hours after the transfection of pGE-HBVP2, the strongest inhibitor among the five, the inhibitory rates of HBsAg secretion in the supernatant were 28.88%, 32.28%, 29.10%, and 18.42% respectively; and the inhibitory rates of HBeAg secretion were 38.33%, 27.50%, 33.41%, and 12.60% respectively. In view of the transfection efficiency of 30%-40%, the actual inhibitory rate of HBV antigen secretion might reach 80% and over. 24 hours after the transfection the expression rate of HBsAg in the 2.2.15 cells transfected with pGE-HBVP2 was 50%, significantly lower than that in the cells transfected with the blank vector pGE-1 (82%). CONCLUSION: siRNAs targeting the HBV P gene effectively prevent the HBV gene expression and replication and may play an important role in the clinical anti-viral treatment.

Bacteriocin Plasmids↗
PubMed2003

[Potent and specific inhibition of hepatitis B virus antigen expression by RNA interference].

OBJECTIVE: To develop an RNAi approach that specifically targets the core gene sequence of hepatitis B virus by synthesizing short interfering RNA (siRNA) in vivo, and to assess the inhibitory effect of this siRNA on HBV replication. METHODS: The eukaryotic expression plasmid pHBV1.3, which contains 1.3-fold-overlength genome of HBV, were cotransfected into HepG2 cells with either the RNAi plasmid pSIHBV/C or unrelated control plasmid pSI. At 24, 48, 72 hours post transfection (p.t.), the levels of HBsAg and HBeAg in the cell culture medium were determined with Abbott MEIA Kits. The expression of intracellular viral proteins was also determined by immunofluorescence staining. RESULTS: Successfully constructed expressing siRNA vector pSIHBV which targeted the core gene of Hepatitis B virus.The introduction of RNAi plasmid was shown to efficiently and specifically inhibit the synthesis of surface and e antigen of HBV by Abbot MEIA kits, with inhibitory rates at 92%, 85% peaking at 24 hours p.t. Immunofluorescence staining showed that intracellular synthesis of viral antigen was sharply reduced to nearly background levels when the ratio of pSIHBV/C and pHBV1.3 was at 1:20, whereas the control vector did not exhibit any inhibitory effect on the replication and expression of HBV. CONCLUSION: Our results demonstrate that the short interfering RNA targeting HBV core gene exerts robust inhibition on HBV viral replication, suggesting that RNAi-based anti-HBV replication strategy may represent a potentially efficacious approach to the clinical management of HBV infection.

Fluorescent Antibody Technique↗
PubMed2006

Dissecting self-renewal in stem cells with RNA interference.

We present an integrated approach to identify genetic mechanisms that control self-renewal in mouse embryonic stem cells. We use short hairpin RNA (shRNA) loss-of-function techniques to downregulate a set of gene products whose expression patterns suggest self-renewal regulatory functions. We focus on transcriptional regulators and identify seven genes for which shRNA-mediated depletion negatively affects self-renewal, including four genes with previously unrecognized roles in self-renewal. Perturbations of these gene products are combined with dynamic, global analyses of gene expression. Our studies suggest specific biological roles for these molecules and reveal the complexity of cell fate regulation in embryonic stem cells.

Animals↗
PubMed2000

Combinatorial RNA interference indicates GLH-4 can compensate for GLH-1; these two P granule components are critical for fertility in C. elegans.

We report that four putative germline RNA helicases, GLHs, are components of the germline-specific P granules in Caenorhabditis elegans. GLH-3 and GLH-4, newly discovered, belong to a multi-gene glh family. Although GLHs are homologous to Drosophila VASA, a polar granule component necessary for oogenesis and embryonic pattern formation, the GLHs are distinguished by containing multiple CCHC zinc fingers. RNA-mediated interference (RNAi) reveals the GLHs are critical for oogenesis. By RNAi at 20 degrees C, when either loss of GLH-1 or GLH-4 alone has no effect, loss of both GLH-1 and GLH-4 results in 97% sterility in the glh-1/4(RNAi) offspring of injected hermaphrodites. glh-1/4(RNAi) germlines are under-proliferated and are without oocytes. glh-1/4(RNAi) animals produce sperm; however, spermatogenesis is delayed and the sperm are defective. P granules are still present in glh-1/4(RNAi) sterile worms as revealed with antibodies against the remaining GLH-2 and GLH-3 proteins, indicating the GLHs function independently in P granule assembly. These studies reveal that C.elegans can use GLH-1 or GLH-4 to promote germline development.

Amino Acid Sequence↗
PubMed2000

Functional genomic analysis of C. elegans chromosome I by systematic RNA interference.

Complete genomic sequence is known for two multicellular eukaryotes, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, and it will soon be known for humans. However, biological function has been assigned to only a small proportion of the predicted genes in any animal. Here we have used RNA-mediated interference (RNAi) to target nearly 90% of predicted genes on C. elegans chromosome I by feeding worms with bacteria that express double-stranded RNA. We have assigned function to 13.9% of the genes analysed, increasing the number of sequenced genes with known phenotypes on chromosome I from 70 to 378. Although most genes with sterile or embryonic lethal RNAi phenotypes are involved in basal cell metabolism, many genes giving post-embryonic phenotypes have conserved sequences but unknown function. In addition, conserved genes are significantly more likely to have an RNAi phenotype than are genes with no conservation. We have constructed a reusable library of bacterial clones that will permit unlimited RNAi screens in the future; this should help develop a more complete view of the relationships between the genome, gene function and the environment.

Animals↗