siRNA to shRNA - (Jun/09/2005 )

Hello everyone,

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 ?

Thanks

David

hi
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.

Good luck.

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 !

Thanks

David

Hi David,

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.

Hi Pcrman,

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...

David

>>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...

Thanks anyway

David