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Five Ways to Produce siRNAs

owever, relies on in vivo transcription of siRNAs from DNA templates introduced into cells. One advantage of these two approaches is that there is no need to work directly with RNA.

Method #4: siRNA Expression Vectors
Most siRNA expression vectors rely on an RNA polymerase III (pol III) promoter to drive the expression of a small hairpin siRNA in mammalian cells (14). RNA pol III was chosen to drive siRNA expression because it naturally expresses relatively large amounts of small RNAs in mammalian cells, it terminates transcription upon incorporating a string of 1-4 uridines, and its transcripts lack poly(A) tails.

To use siRNA expression vectors, two oligodeoxynucleotides encoding the desired short hairpin RNA sequence are ordered, annealed, and cloned into the vector downstream of the promoter. Because cloning is involved, the procedure takes several days, and sequencing the region containing the insert is required. However, this limitation is balanced by the ability to produce large quantities of vector once the vector is shown to work well in gene silencing experiments.

Without a question, the main advantage of siRNA expression vectors is that they are amenable to long term studies. Vectors with antibiotic resistance markers can be used to reduce the expression of targeted genes for several weeks or longer. Transient selection of cells transfected with selectable marker containing plasmids also permits the enrichment of cell
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