siRNA to shRNA - (Jun/09/2005 )
I have a siRNA duplex that works well and I would like to cut on the cost of reorders and clone this siRNA into a shRNA vector.
In fact, I'm not sure if I can do that but if it is possible, is there anybody here who tried and succeeded to do it ?
there should be no problem to clone your siRNA in a shRNA expression vector. You just need to add a loop between your sequences (sense and antisense). Little bibliography would give you more informations. I can recommend you this sequence that works well :
TTCAAGAGA from Brümmelkamp and al. • Science 296, 550-553
Vectors are commercially available and H1 / U6 promoters are quite equivalent to ensure good transcription.
I would add an other recommendation : if you plan to buy a vector, i'd rather choose one with puromycin resstance gene instead of neomycine. Neomycine is very less interesting as puromycin regarding cell selection procedure.
Yes I have done it. I cloned my working siRNA into pSilencer neo vector from Ambion by ordering two oligos, annealing them, ligating into the vector. Everything went smoothly and the results are expected.
pcrman, do I understand you didn't use any loop between your sense and antisense strand ?
I wonder if this system is as efficiency as the hairpin system.
I have to say that I'd rather prefer your way as I don't need to spend time on designing and synthetizing a loop...I just want to put my working duplex in a vector and go on !
The loop sequence is included in the oligos which are 55 nt and every shRNA needs it. Also incoporated in the oligos is a restrictin site compatible with the stick end of the vector. Please check Ambion's manual for oligo design. It may take a while to figure out.
Did you use two oligos strictly complementary ? I've heard from a neighbour lab that i should introduce one or two mutations in one of the two oligos to avoid difficulties during the cloning into the vector.
Have you ever heard of that ?
Thank you for your answer...
>>Did you use two oligos strictly complementary ? I've heard from a neighbour lab that i should introduce one or two mutations in one of the two oligos to avoid difficulties during the cloning into the vector. Have you ever heard of that ?
No. I just follow Ambion's instruction and everything went smoothly. I have sequenced my plasmid, lucky enough, every clone I picked has the right insert. My oligo annealing, ligation and transformation, etc all behaved as expected.
Ambion also has a shRNA design tool, you just input the target seqence it will give you the oligos with restriction ends, loop incorporated. It save you lot of pain and mistakes.
Thanks a lot for your answer...
In fact, I wish I could follow your advice but my boss want me to borrow a vector used by a neighbour lab and use the sequence of the RNAi we tested some weeks ago. I spent a lot of time to set the conditions right and now that it is working fine, I have to restart from the beginning with shRNA instead of simply reordering the RNAi oligos. So I have to design a shRNA by myself with the RNAi sequences that works and add the loop and the restriction sites and of course re-define the conditions again !
I'm a little upset about this...