Journal of RNAi and gene silencing : an international journal of RNA and gene targeting research最新文献

Introduction of small interfering RNAs (siRNAs) into cells results in transitory silencing of target genes with complementary sequence. Incorporating siRNAs into short-hairpin RNAs (shRNAs) or microRNA-adapted shRNAs (shRNAmir) is a popular tool for targeted gene silencing. shRNAmirs mimicking endogenous pre-microRNAs (unprocessed hairpin microRNAs) are more difficult to design and result in longer RNA molecules. The use of microRNA (miRNA) loop sequences in shRNAs as an alternative to an entire pre-microRNA structure on silencing efficiency has not been studied extensively. This report shows that loop sequences derived from native miRNAs improves the efficiency of silencing due to the processing of the shRNAs into mature siRNAs.

c-Kit, the gene product of the W locus is a receptor tyrosine kinase that regulates the survival, growth and differentiation of spermatogonial cells (SGCs). Stem cell factor (SCF), the gene product of the steel (Sl) locus is the ligand for c-kit. Normal function of SGCs requires cross-talk between c-kit and SCF through which the receptor-ligand pair regulates the functions of SGCs. The implications of cross-talk between c-kit and SCF in regulating SGC function remains unclear due to the molecular complexity of this interaction. In the present study, we analyzed the interactions between c-kit and SCF in mouse primary SGCs after blocking the c-kit expression by c-kit siRNA and its effect on cell fate were determined using cDNA Expression Array and Real-time PCR. Immunofluorescence (IF) and western blot studies revealed that c-kit protein was detected in SGCs and knocked down to undetectable levels at 24 hr post transfection with 10 nM concentration of c-kit siRNA. We further demonstrated that expression of various genes involved in cell signaling, cell differentiation, apoptosis and cell cycle pathways was altered. SGC functions are affected by SCF signaling through c-kit receptor and this signaling appears to be important to maintain balance between cell proliferation and apoptosis along with the modulation of inflammatory responses of SGCs. To the best of our knowledge, this is the first report that identifies the putative molecular pathways in murine SGCs in response to specific blocking of c-kit-SCF interactions by siRNA. In conclusion, the present study may provide useful insights into siRNA function and hopefully aid in understanding the involvement of c-kit in the early events of SGC activities and spermatogenesis in mice.

RNA interference-mediated gene silencing offers the potential of targeted inhibition of disease-relevant genes. In vivo delivery of RNAi reagents can be obtained by a variety of approaches. Physical delivery methods appear safer and lack side effects. Electro-permeabilization is one of the non-viral methods successfully used to transfer small interfering RNAs (siRNAs) in vitro and in vivo. A promising approach may be, very little is known about the fundamental processes mediating siRNA transfer. In this study, we have investigated cellular delivery pathways involved in electro-delivery of siRNAs by a direct fluorescence imaging method. An Alexa-labeled siRNA was electro-transferred into murine melanoma cells stably-expressing the enhanced green fluorescent protein (eGFP) target reporter gene. The silencing of eGFP gene expression was quantified by time-lapsed fluorescence microscopy. Fluorescently-labeled siRNAs were found distributed homogeneously in cytoplasm 48 hours after electro-transfer, apparently by diffusion. Furthermore, siRNAs showed homogeneous distribution in vivo 48 hrs after intra-tumoral injection followed by electro- permeabilization. Histological fluorescence microscopy showed that siRNAs were mostly localized in the cytoplasm. Overall, this study shows that electro-permeabilization facilitates cytoplasmic distribution of siRNA, both in cultured cells and in vivo. This method offers a potential therapeutic tool to facilitate direct siRNA penetration into solid tumors.

The RNA polymerase III (pol III) type III promoters U6 and 7SK are routinely used to express short hairpin RNA (shRNA) molecules from a DNA construct. In this study, we identified, characterised and compared the porcine 7SK promoter in porcine (homologous) and non-porcine (heterologous) derived cell lines. The porcine 7SK small nuclear RNA (snRNA) was identified by alignment with known sequences and further characterisation of the upstream regions determined the presence of typical RNA pol III sequence motifs. The porcine 7SK (po7SK) promoter was cloned and a one-step PCR strategy used to construct shRNA expression cassettes. The 7SK promoter activity was quantified by knockdown of the exogenous reporter gene encoding the enhanced green fluorescent protein (EGFP). Results indicated the po7SK promoter was functional in both homologous and heterologous cells lines. The identification and characterisation of the porcine RNA pol III promoter will contribute to the area of RNAi delivery and further develop our understanding of RNA promoter structure and function.

RNAi can mediate allele-specific silencing, and offers an attractive approach for treatment of human diseases caused by dominant, gain-of-function gene mutations. However, allele-specific silencing requires that the RNAi target the mutated region of the mRNA, limiting the choices of the target sequences. This often results in the use of a suboptimal siRNAs or shRNAs. Unfavorable strand asymmetry, which leads to the sense strand rather than the antisense strand to be incorporated into RNA-induced silencing complex (RISC), can cause poor RNAi efficacy. We systematically tested an approach that places mismatches at or near the 5' of the antisense strand to create favorable strand asymmetry. Here we show that this approach can enhance the RNAi efficacy of not only siRNAs but also shRNAs synthesized from genes, which can be placed in various viral vectors. Thus, this design of asymmetric shRNAs could be potentially used in silencing dominant, gain-of-function gene mutations for gene therapy.

Recently, artificial microRNA (miRNA)-mediated RNA interference (RNAi) systems have been developed as useful tools to study gene functions. We report an artificial miRNA precursor motif (AMPM) containing several restriction sites in the loop and in the extended stem structures, and generated a vector-based expression system for AMPM under the control of an RNA polymerase II promoter. The AMPM located in the intron or the exon of the selection marker gene mediated silencing of the reporter luciferase gene expression in HeLa cells. Northern blotting and colony formation assays revealed that the AMPM was efficiently and appropriately processed into mature miRNA. The AMPA system also down-regulated endogenous p53 and laminA/C proteins in stable transfectants. Moreover, clustered AMPMs separated by an appropriate spacer, targeting different sites within a single mRNA mediated gene silencing, even if the sequences of the target and the AMPM were partially complementary. This indicates the potential therapeutic utility of clustered AMPMs for highly-mutable targets, such as human immunodeficiency virus type 1.

The application of RNA interference (RNAi) to study gene function is now commonplace in a variety of biological systems. Producing short hairpin RNA (shRNA) by DNA vectors is one popular strategy for RNAi applications. Here, we describe a one-step PCR method, termed reverse PCR, for constructing shRNA expression vectors. Characteristically, the pair of primers binds to circular plasmid in a back-to-back manner. The anchored primers provide the templates of shRNA sense strand and antisense strand locating to the two separate ends of PCR segment, which will benefit the PCR amplification and subsequent cloning by avoiding premature formation of a hairpin configuration. Finally, the establishment of a circular vector is achieved by self-ligation of the single PCR product. In addition, our results indicated that the hairpin loop including a single restriction site is resistant to digestion, while inclusion of twin restriction sites in the loop leads to activity, creating an optimal strategy for verifying sequences of shRNA template.