A PCR-based strategy for cloning short hairpin sequences:“PCR shagging”.Our overall approach is to use an RNA polymerase III promoter to driveexpression of encoded short hairpin RNA (shRNA). For this purpose we use the U6snRNA promoter and maintain the transcript initiating “G” nucleotide of the U6snRNAtranscript. There by, hairpin sequences will start with a “G”. Termination is mediatedby a run of Ts at the end of the hairpin.The major difference between our hairpins those reported by others is that wewent through a battery of tests of hairpin length and structure, and found that hairpins of27 to 29nt in length are more effective than hairpins of 19nt and 21nt. Additionally, weuse 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 shRNAsequences. In this strategy, short hairpin RNA (shRNA) sequences are converted into asingle ~73nt primer sequences onto which are added 21nt of homology to the human U6snRNA promoter. Such primers have performed flawlessly so far in PCR reactions(n>40) and subsequent cloning.PCRTA/TopoCloningHindIII siteTransient transfection Subcloning (eg Invitrogen’s Gatewaysystem)U6 promoterU6 promoter TsU6 promoter TsThere are several steps in generating hairpin primers. First, a 29nt “sense” sequencewhich ends with a “C” is picked out from the coding sequence of gene of interest.Second, the actual hairpin is constructed in a 5’->3 orientation with respect to theintended transcript.Anti-sense Loop Sense TermggctatgaagagatacgccctggttccGaagcttGggaaccagggcgtatctcttcatagccTTTTTTGPredicted shRNA structure5’->3’ Anti-sense strand-------| GAAGGCUAUGAAGAGAUACGCCCUGGUUCC GCCGAUACUUCUCUAUGCGGGACCAAGG CUU^ GUU3’<-5’ Sense strandThird, a few stem pairing are changed to G-U by altering the sense strand sequence. G-Ubase pairing seems to be essential for stability of short hairpins in bacteria and doesnot interfere with silencing. Finally, the hairpin construct is converted to its “reversecomplement” onto which is added 21nt of homology to the Human U6 promoter.Hairpin portion of the primer (~69nt)CAAAAAAggctatgaagagaCacgccctgAttccCaagcttCggaaccagggcgtatctcttcatagcc+U6 promoter reverse primer sequenceGgt gtt tcg tcc ttt cca caaFinal primer (5’->3’, just as it would be ordered)CAAAAAAggctatgaagagaCacgccctgAttccCaagcttCggaaccagggcgtatctcttcatagccGgt gtt tcg tcc ttt cca caaAll of the aforementioned steps are automated using a program developed by RaviSachidanandam and Jeremiah Faith (CSHL) where either accession numbers fromGenBank or raw sequences are required to generate hairpin PCR primers.[Note: Don’t let the G-U pairings represented in the primer fool you into thinkingthe primer is incorrect. ]A link to the hairpin primer generation program, the “RNAi oligo retriever”, can befound at:www.cshl.org/public/SCIENCE/hannon.htmlMake sure that you enter accession numbers and sequences which match cDNA orexon sequences!
THE PROTOCOLOrdering PrimersSince very little primer is required for the PCR reaction they can be ordered .05μmolscale from Sigma-Genosys or whomever. We find that PAGE purification is costly andunnecessary (PCR will fill in shorted primers!).PCRWe use a pGEM1 plasmid containing the human U6 locus (N. Hernandez, CSHL) as thetemplate for the PCR reaction. This vector contains ~500bp of upstream U6 promotersequence. Since an SP6 sequence flanks the upstream portion of the U6 promoter, weuse an SP6 oligo as the universal primer in U6-hairpin PCR reactions.SP6 sequence: GATTTAGGTGACACTATAGWe have had consistently good results using Taq polymerase for PCR with 4% DMSOand 50pmoles of each primers. (For pENTR/D-Topo cloning [see below], I add .1uLof Vent to polish the ends.)PCR conditions: 95° for 3 min; 30 cycles of 95° for 30 sec, 55° for 30 sec, & 72° for 1min; followed by one cycle of 72° for 10 min.The PCR product will be ~600bp in length.CLONINGWe currently use two cloning technologies available from Invitrogen: T-A anddirectional topoisomerase-mediated cloning kits (catalog #K2040-10, K2400-20). Thedirectional cloning kit is designed for Invitrogen’s Gateway system. We use both kitsaccording to the manufacture’s instructions. If using Topo-cloning, do NOT gel purifyPCR products – it reduces the efficiency of the Topo-reaction.pENTR/D-Topo SP6 primer: CACC GATTTAGGTGACACTATAGFor convenient identification of clones containing the proper insert (20-100% for Topocloning),a HindIII site has been designed into the loop of the hairpin. A second HindIIIsite exists 5’ of U6 promoter. Digesting clones with HindIII releases a ~500bp fragment.SP6-U6 promoter PCR product sequence (with out hairpin)*.SP6—HindIII—BamHI—U6 promoter