PSuper RNAi System
For retroviral shRNA mediated knockdown we use the pSUPER RNAi system from OligoEngine.
- BglII and HindIII (or alternatively XhoI. See manual on OligoEngine website) restriction enzymes
- pSuper/pSuperior vector (oligoEngine)
- Competent cells
- HEPES pH 7.4
- 100mM NaCl
- 50mM HEPES pH 7.4
To design the shRNA oligos targeting specific mRNAs, we use the siRNA design tool available on the Dharmacon website. The tool returns a number of possible siRNAs sorted by score from the most likely to the least likely to knock down the protein.
Some fields of interest to note in the table are:
- Low Seed Freq?: Yes/No indicator of a low siRNA seed region frequency within the 3'UTR of the target (or host) genome. siRNA with low frequency seed regions will have reduced off-target effects relative to higher frequencies. (Preferred: Yes)
- Requires Sense Mods?: Yes/No indicator of whether the siRNA design should be synthesized with a chemical modification to inhibit sense-strand uptake. (Preferred: No)
- Min # mismatches (sense/antisense): The lowest number of mismatches of the 19-base antisense strand to a sequence record other than the required target(s) as determined by BLAST analysis. (Preferred: Higher numbers)
More information about this tool can be found in the online user guide.
We usually select 3-5 oligos from the list and then screen them for optimal knockdown. Once the oligos are chosen, proper adapters must be added to the sequence. Two sequences are created (sense and antisense). HindIII version is shown.
- Sense: 5'-GATCCCC<oligo>TTCAAGAGA<rev. compl. of oligo>TTTTTA-3'
- Antisense: 5'-AGCTTAAAAA<oligo>TCTCTTGAA<rev. compl. of oligo>GGG-3'
Creating these oligos can be automated by using the Gen shRNA tool on our website. The bookmarklet integrates this tool into the Dharmacon website.
The oligos can now be ordered.
- Resuspend the oligos in H2O at 3mg/ml
- Mix 1µl of each oligo with 48µl of annealing buffer
- Incubate the mixture at 90C for 4 minutes then 70C for 10 minutes
- Slowly cool the oligos to 10C (e.g. step-cool to 37C for 15-20 mins, then cool to 10C) and move to 4C
A thermal cycler can be programmed to make this process automatic.
- Linearize the vector of choice using BglII and HindIII (or XhoI) for 60 minutes.
- Gel-purify the vector on a 1% agarose gel. The vector should run at about 6300kb.
- Resuspend the vector to a concentration of 0.2-0.5mg/ml
- Ligate 2µl of annealed oligos and 1µl of linearized vector using T4 DNA ligase overnight at room temperature.
- Add 5 units of BglII to the ligation reaction and incubate for 30 minutes at 37C.
- Ligating the annealed oligo into the vector destroys the BglII site. This will get rid of vectors that were only cut once and self-ligated.
- CAUTION: if your hairpin oligo contains a BglII site, SKIP THIS STEP.
- 6. Transform the vector into bacteria and plate it on Amp media.
- Prepare minipreps of the ligated vectors.
- Digest with EcoRI and HindIII.
- A negative clone will have a band at ~227bp
- A positive clone will have a band at ~281bp
- 3. Send selected clones for sequencing. Almost all of our clones generally come back with correct sequences.
Determining optimal KD
Follow the retroviral packaging and spinfection protocol and check knockdown levels for each hairpin. The best knockdown is generally what you want to use. Knockdown specificity should be verified by rescue with siRNA-immune construct of the gene. Using two different siRNAs to show the KD specificity is also recommended.