Best Vector for shRNA production - Help (Oct/25/2005 )
Hello
I'm thinking about convincing my supervisor to try out an shRNA vector for knockdown of our protein of interest. Anyone know which vectors seem to work the best in your hands?
I've heard good things about the pSUPER vectors with the H1 promoter. Any others?
How much work am I taking on really to start designing an shRNA vector in terms of time?
Thanks, any advice would be greatly appreciated to an RNAi rookie,
Mountainman 
hi
there is now many valuable vecto, you've mentionned pSUPER, but you have also pSILENCER which is great... You should take in care
which promoter you want U6/H1 but these two have prooven their efficiency.
which selection you want : neo, puro, blasticidin, zeocin... this point is important if your cell have already a resistance, or if you want to use multiple resistance...
which way you want to deliver : trnasfection or retrovirus...
designing a shRNA can take several month if you have not already a sequence. For help on tat, you have many online tools that can help. You've also on the forum related to RNAi a database on validated RNAi sequences.
Do you guys know that The RNAi Consortium provides cloned shRNA. You can search from their database and if you find your gene, you can order from Sigma in the form of bacterial glycerol stock, Plasmid DNA or transduction particles. This will save you lots of time and efforts constructing your own shRNA.
Hey Thanks,
It's good to know these things before I start cloning. Are the shRNA's from the RNAi consortium validated for knockdown? I've heard of people having to try many before one finally works.
Thanks again
Mountainman
Has anyone tried pRNAt-H1 vectors from Genescript? They have a GFP marker for detection which I think would be quite usefull.
However this is not present in the pSilencer constructs, therefore how do you detect transfection ( and rates of transfection) with pSilencer?
However this is not present in the pSilencer constructs, therefore how do you detect transfection ( and rates of transfection) with pSilencer?
I am using it right now
it comes with pmaxGFP positive control that can be used for checking transfection efficiency
u can also digest the expression casette using I-CEU-1 AND pSCE-1 RESTRICTION ENZYMES TO TRANSFER IT INTO P-ADENOx FOR INVIVO STUDIES