need advice on suppliers!

[Roselyn]

Hi all,

I'm new to the wonderful world of gene suppression - I have human primary cells (fibroblasts) and am trying to knock down 3 subtypes of a G protein-coupled receptor to dissect their individual contributions in our system.

Does anyone out there recommend a particular way to go about silencing a gene that has very high expression? One of the subtypes does; I started with morpholinos but that is not doing the trick and am considering Clontech's Knockout single vector system for inducible RNAi; Superarray's plasmid-based SureSilencing shRNA;Dharmacon's product or Origene's HuSH-29 shRNA (packaging into a retrovirus). I am leaning toward's Origene's product. I have a small grant with limited supply$$ so I need to get it as right as I can the first time around! Any advice for me? Words of experience and guidance will be much appreciated!

[moleculardude]

Hey there Roselyn,

the best way to silence a gene is by knocking it out. Since you probably cant do that, the best other way is to integrate a plasmid containing a very good siRNA/RNAi sequence that expresses it highly.

Unfortunately, you need to test out several RNAis to do this, as you dont want to go through the trouble of cloning your RNAi and making a stable cell line of it....

I would look for companies that guarantee results (ie their RNAi works), although this may be tough to find and more expensive.

The cheapest way would be to design a few RNAi's yourself, and then borrow (i didnt say that! ) or purchase the RNAi vector to make stables

(it should have Neo cassette or Zeo)

cheers