siRNA "knock-out"/transfection "knock-in" - (Dec/30/2007 )
I'm studying this cytoplasmic protein that is ubiquitously expressed in all cell lines my lab works with (HEK293 and 293T and HeLa and RAW and A549 and CHO and...). However, for some experiments I would need this protein to be absent from the cells so that I could study the effect that its mutant forms have.
Since I don't have access to KO cell lines, there really is no easy way to get round this. So I thought maybe I could try doing the following: I would »knock out« this endogenous protein via silencing and then »knock-in« the desired mutated form of this same protein again via transfection... Basically it would be like some kind of rescue control/resistant gene.. Do you know of any siRNA companies that would simultaneously synthesize the siRNA and this "resistant" gene?
Do you guys think that's even possible? Or is it really just a stupid idea? I actually have no experience working with siRNA. I imagine I should be worried about the level of the silencing as well?
Thanks a million for your thoughts on the subject!
Transient co-transfection with an siRNA agianst your endogenous mRNA and an expression vector with mutant gene would be the easiest way to achieve that, particularly if your mutant gene is a truncated mutant and siRNA is agaisnt the 5' untranslated region or the truncated region that your expression vector does not carry either region. Companies can help you to do so, but may not tell you the sequences. There are softwares you can use to design siRNA by yourself, or oftentime you can find such info from publications.
well for doing this on my project, i would suggest you following ways.
first try to generate stable cell lines expressing your protein of interest. the best is to use an expression vector which drives expression of an epitope tagged protein (for further easy coIP assays)
second you designate 3 siRNAs against your protein and test them on both wt cell line and expressing one
you see there if your siRNAs are spécific & efficient, and you have first idea about your mutant form role
if you plan long term silencing, use expression of shRNA. You can use H1 U6 or 7SK promoters for that.
Best is to use pSUPERIOOR vectors (oligoengine) in which expression of siRNA is inductible by tetracycline.
Remember, that knocking down with siRNA, you will not get 100% knockdown, there could be still be some effect from the remaining protein. You will need to verify if this is fine for your experiments.
Thanks for your output guys!
Hey fred, I'm not sure I understand what you mean by "designate 3 siRNAs". Why would I need 3 of them?
And yes, scolix, I am a bit concerned about the level of the silencing I can ahieve this way, but I don't see any other way of approaching this problem at the moment.
Because not every design works.
you should be lucky to have a good one among 3. We design more than 3 to test knockdown.
before you get too fancy designing a construct that can't be targeted with your siRNA, often you can just use, say, the mouse gene for overexpression if you are targeting the human with siRNA. You may already have the mouse gene in your lab who knows.
May save you some time if it work
May save you some time if it work
Huh, somehow I haven't thought of this! Thanks for the idea. Although the protein is pretty conserved - there's much sequence similarity. But yeah, sure, it's something worth considering!