Designing shRNA - (Dec/11/2008 )
I would like to down-regulate a gene by using siRNA in vivo.
We will use our own retroviral vector.
However, it is not clear where the transcription starts and ends with our vector which has the multiple cloning site between SV40 promoter and polyA sites.
My guess is that the siRNA will have long 5' end and 3' end in addition to the target stem loop.
Does this matter or it will be processed okay in vivo.
For shRNA cloning, you don't need to add extra sequence up- and down-stream the target sequence, but you need to carefully select the loop sequence. For miRNA cloning, 100-200 bp sequence is usually added to the pre-miR sequence.
Thanks for the insights.
I am trying to design siRNA under pgk promoter on my retroviral vector.
Even though I don't add extra sequence on my target sequence as you suggest, I assume I will have extra sequence on the upstream side, because there are still unwated sequences between the transcription +1 site and the cloning site.
I do not know where transcription +1 site is.
My educated guess is about 50nt upstream of the cloning site which means that I will have at least 50nt upstream sequence besides my siRNA target sequence.