Are there any unique elements that need to be incorporated into an shRNA expression vector?
For most RNAi applications, little shRNA expression is needed, because RNA interference is a catalytic process. The over-expression of an shRNA may induce off-target effects and stimulate toxic side effects such as the interferon response and apoptosis. In order to avoid these problems, shRNA expression plasmids are often engineered such that expression is regulated by a relatively weak promoter. For example, the SureSilencing shRNA Plasmids express shRNA from the U1 promoter. There have also been reports of successfully using RNA polymerase III promoters to control shRNA expression. When these promoters are used, the sequence downstream of the shRNA is devoid of a polyadenylation signal. Such transcripts are not extensively processed on their 5’ and 3’ ends, and are reported to serve as better substrates for the Dicer ribonuclease. With the exception of the promoter sequence modifications, the remaining sequences within a shRNA expression plasmid are typically the same as those found in traditional mammalian cell expression vectors.