Our overall approach is to use an RNA polymerase III promoter to drive expression of encoded short hairpin RNA (shRNA). For this purpose we use the U6 snRNA promoter and maintain the transcript initiating “G” nucleotide of the U6snRNA transcript. There by, hairpin sequences will start with a “G”. Termination is mediated by a run of Ts at the end of the hairpin.
The major difference between our hairpins those reported by others is that we went through a battery of tests of hairpin length and structure, and found that hairpins of 27 to 29nt in length are more effective than hairpins of 19nt and 21nt. Additionally, we use a few G-U pairing in the hairpin stem (which are permitted in dsRNA alpha helices) to stabilize hairpins during propogation in bacteria.
We have developed a fast and effective, PCR-based strategy to clone shRNA sequences. In this strategy, short hairpin RNA (shRNA) sequences are converted into a single ~73nt primer sequences onto which are added 21nt of homology to the human U6 snRNA promoter. Such primers have performed flawlessly so far in PCR reactions (n>40) and subsequent cloning.
