siRNA stable cell lines - (Aug/15/2007 )

Hello,

Is it possible to create a stable cell line with siRNA for a specific gene. ONce this is established can a stable cell line overexpressing another gene be created. Therefore, can you make a second stable cell line from an already created cell line?

thanks

Hi,

yes, you can create stable 'knock-down' clones by expressing shRNA (pSuper.neo is a widely used system for example). You can select with G418 I think.
Once these clones are established you can also transfect the stable clones a second time - the selection marker needs to be different from the first one.
However, I think it all depends on the cells you are working with, some will make it, others will not. I'm afraid it is not easy and a lot of work.

Cheers

well, for establishing a siRNA expressing cell line, i would go for a puro selection marker. it's so quicker than G418, and you're definitely sure of your cells.
For expressing a protein, as you neeed to establish cell clones, i like in this situation the G418. Clones are sufficently diluted to ensure good G418 selection, and as you check some cell clones for good expression of your protein you have a second check.

The trick is that you neeed 2 different selection markers, and they need to be different as the selection marker which may have been used for cell line establishment.

Once I establish the siRNA expressing cell line for protein X, can I overexpress protein X or will protein x be silence because my cell line is now an siRNA expressing cell line??

Thanks

well, that is supposed to happen.
E.g. Amaxa suggests to transfect a GFP vector and a GFP vector plus one GFP specific siRNA into cells to have a control about experimental settings.
In this case it is just transient.

In a stable situation I would also suppose to see a less expressed protein X in cells expressing a protein x specific siRNA compared to cells with a non protein x specific siRNA.

Unless you really are in bad need for such cells for whatever reason I cannot imagine, it seems pretty much work to get such cells finally (in the sense of: not just having them selected but also characterized and compared).
Usually, the aim is to get more than one clone to be able to set up experiments in triplicate or whatever... .
You would also need negativ control clones e.g. cells that overexpress the protein x and a shRNA vector (whether not targeting 'anything' in the cell or an siRNA at least not specific to protein x).

Cheers

I have pSuper basic vector and there is no selection marker to generate stable cell line. Did you try to co-transfect with a plasmid containing antibiotic resistence gene and then make stable knock-down. Does it work? or is there anybody to send me antibiotic resistence containing plasmid.
thx