Help needed with shRNA interference....

[Simon Convey]

Dear DIYbio,

        I've reverse engineered a short hairpin RNA sequence that produces an interfering RNA that I want to express in human cells. 

This RNAi targets myotilin, which when misfolded causes a very rare form of muscular dystrophy. Eliminating the misfolded myotilin largely reverses the disease symptoms in both mice and rhesus monkeys. No side effects from the loss of myotilin were observed.

         I now have no idea how to proceed with getting AAV to express the above. The design, and manipulation of plasmids is beyond me, so I'm happy to ask a plasmid factory to do this for me, it's just that I don't know what to ask for......

Any help would be appreciated.....

Many thanks,

Simon.

[Andreas "Mega" Stuermer]

human U6 promoter has a defined beginning and ends in TTTT so people use it a lot. h1 Promoter too. (Pol III promoter vs Pol II promoters). If you want to be very fancy, add ribozymes to cut off the 3' extension or any 5' extension. 

Some people have also used tRNA as promoter and cutting signals, and it didn't really seem to mess up cellular metabolism. 

Let me know if you have any further questions

[James Welch]

This paper seems pertienent to what you want to accomplish. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4460833/ . Additionally, googling "miRNA vectors" produces some good plasmids which would might be useful for cell culture work. I don't have experience with AAVs but it seems like there are plasmids for purchase which are made for miRNA delivery. Hope that helps.