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Lentiviral delivery of designed shRNAs and the mechanism of RNA interference in mammalian cells. RNA interference (RNAi) is a biological process in which RNA molecules are involved in sequence-specific suppression of gene expression by double-stranded RNA, through translational or transcriptional repression.
The RLC consists of dicer, the transactivating response RNA-binding protein and Argonaute 2. Dicer is an RNase III endonuclease which generates the dsRNA fragments to be loaded that direct RNAi. TRBP is a protein with three double-stranded RNA-binding domains. Argonaute 2 is an RNase and is the catalytic centre of RISC.
RNA silencing describes several mechanistically related pathways which are involved in controlling and regulating gene expression. [5] [6] [7] RNA silencing pathways are associated with the regulatory activity of small non-coding RNAs (approximately 20–30 nucleotides in length) that function as factors involved in inactivating homologous sequences, promoting endonuclease activity ...
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded non-coding RNA molecules, typically 20–24 base pairs in length, similar to microRNA (miRNA), and operating within the RNA interference (RNAi) pathway.
Potential oversaturation of RISC is also a problem. If the shRNA is expressed at levels that are too high, the cell might not be able to correctly process the endogenous RNA which could cause significant problems. Another challenge is the possibility that the patient might mount an immune response against the therapy. [10]
DNA-directed RNA interference (ddRNAi) is a gene-silencing technique that utilizes DNA constructs to activate a cell's endogenous RNA interference (RNAi) pathways. DNA constructs are designed to express self-complementary double-stranded RNAs, typically short-hairpin RNAs (shRNA), that bring about the silencing of a target gene or genes once processed. [1]
RNAI is a non-coding RNA that is an antisense repressor of the replication of some E. coli plasmids, including ColE1. Plasmid replication is usually initiated by RNAII, [1] which acts as a primer by binding to its template DNA. The complementary RNAI binds RNAII prohibiting it from its initiation role.
Gene knockdown is an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.