Biomedical subjects

Vivi Kasim

Publications and source records attributed to Vivi Kasim.

4 recordsLinked to original sources

PubMed2006

Screening of siRNA target sequences by using fragmentized DNA.

BACKGROUND: RNA interference (RNAi) has become a powerful tool in silencing target genes in various organisms. In mammals, RNAi can be induced by using short interfering RNA (siRNA). The efficacy of inducing RNAi in mammalian cells by using siRNA depends very much on the selection of the target sequences. METHODS: We developed an siRNA target sequence selection system by first constructing parallel-type siRNA expression vector libraries carrying siRNA expression fragments originating from fragmentized target genes, and then using a group selection system. For a model system, we constructed parallel-type siRNA expression vector libraries against DsRed and GFP reporter genes. RESULTS: We carried out the first screening of groups containing more than 100 random siRNA expression plasmids in total for each target gene, and successfully obtained target sequences with very strong efficacy. Furthermore, we also obtained some clones that express dsRNAs of various lengths that might induce cytotoxicity. CONCLUSIONS: This system should allow us to perform screening for powerful target sequences, by including all possible target sequences for any gene, even without knowing the whole sequence of the target gene in advance. At the same time, target sequences that should be avoided due to cytotoxicity can be identified.

Base Sequence↗

PubMed2004

Continuous cell partitioning using an aqueous two-phase flow system in microfluidic devices.

We present a novel microfluidic system in which an aqueous two-phase laminar flow is stably formed, and the continuous partitioning of relatively large cells can be performed, eliminating the influence of gravity. In this study, plant cell aggregates whose diameters were 37-96 microm were used as model particles. We first performed cell partitioning using a simple straight microchannel having two inlets and two outlets and examined the effects of the flow rate and the phase width on partitioning efficiency. Second, by using a microchannel with a pinched segment, the partitioning efficiency was successfully improved. This microscale aqueous two-phase flow system can further be incorporated into micro total analysis systems (microTAS) or lab-on-a-chip technology, owing to its simplicity, applicability, and biocompatibility.

Cell Aggregation↗

PubMed2004

Control of siRNA expression using the Cre-loxP recombination system.

Gene silencing mediated by RNA interference (RNAi) was first discovered in Caenorhabditis elegans, and was subsequently recognized in various other organisms. In mammalian cells, RNAi can be induced by small interfering RNAs (siRNAs). In earlier studies, our group developed a vector-based system for expression of siRNA under control of a polymerase III promoter, the U6 promoter, which can induce RNAi in living cells. We here describe a system for controlling the U6 promoter-driven expression of siRNA using the Cre-loxP recombination system. We constructed a 'Cre-On' siRNA expression vector which could be switched on upon excision catalyzed by Cre recombinase, which was delivered to cells directly from the medium as a fusion protein. An examination of the effectiveness of RNAi against a reporter gene revealed that addition of TAT-NLS-Cre (where NLS is a nuclear localization signal and TAT is a peptide of 11 amino acids derived from HIV) to the medium resulted in plasmid recombination, generation of siRNA and suppression of reporter activity. This system should allow us to induce RNAi in a spatially, temporally, cell type-specifically or tissue-specifically controlled manner and potentiate the improved application of RNAi in both an experimental and a therapeutic context.

Amino Acid Sequence↗

PubMed2003

Control of siRNA expression utilizing Cre-loxP recombination system.

Vector-mediated systems for specific siRNA expression in mammalian cells using pol III promoters allowing high level of transcription activity have been developed in the past years, widening the usage of RNA interference (RNAi). In this study, we controlled the pol III promoter (U6 promoter)-driven expression of siRNA using the Cre-loxP system. Our "Cre-On" siRNA-expression vector against firefly luciferase activity could be switched on only in the presence of Cre recombinase, which, in this study, was delivered directly from the medium into the cells as TAT-NLS-Cre, a fusion protein with TAT peptide (an Arg rich peptide derived from HIV) and nuclear localizing signal (NLS). Upon the addition of TAT-NLS-Cre, complete and functional siRNAs were generated and reporter activity was suppressed.

Integrases↗