Hi,
I was wondering if commonly used tagging systems (like FLAG, Streptag or His) can be used as a docking site for knocking down -or even knocking out- tagged proteins
The goal would be to add a third-party molecule/fusion protein that, upon recognition of the tag, could deplete the tagged protein.
Thus, we could have a potential conditionnal KO model by repurposing old/unused transgenic model/cell lines.
I read some stuff about targeted proteolysis and degron systems for example, but i haven't find anything that could be used with common tags.
I'm thinking about using it in mamal primary cell culture or human transformed celle lines.
Does anyone have an idea about it ?
Thanks,
Pierre-Louis
I suppose you could do knockdown via shRNA or siRNA. Or you could try a CRISPR type approach. Or maybe use a PROTACS like approach - express the target antibody intracellularly, modified with a ubiquitination signal?
That being said, it sounds like you want something inducible with minimal chemical treatment. To do that, you'd have to take your tag-recognizing protein, express it in an inducible vector, isolate stable cells, etc. Seems like it'd be easier to just make new cells with your gene of interest in an inducible vector, so that you switched THAT gene on and off, rather than a gene meant to interfere with it. Probably give you cleaner results, and would take exactly the same amount of time and effort.
Hi everyone, thanks for your responses
Yeah I know that there are now efficient & elegants methods to disrupt proteins in a conditionnal way, it seems to work quite well on transformed cell lines or primary human cells.
My point was to ask if rodent model of tag-expressing specific protein could be
« recycled » into a knock-out model for short-term ex-vivo culture .
Like we'd only need to add proteins able to specificly bind the tag like antibodies, fusion proteins or whatever to the culture media (and probably electroporate cells) to deplete the tagged protein at the post-translationnal level.
Thus, we wouldn't need to generate a new KO model. I couldn't find any validated approach doing, as it now seems that it doesn't exist.
Thanks again for your responses,
Pierre-Louis
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