Stable transfection in suspension cells

Ozan Aygun via methods%40net.bio.net (by metugenetics from yahoo.com)
Sun Jan 20 14:11:10 EST 2008


Hi,

This may depend on the suspension cell line that you
are using. For example, although HeLa S3 cells grow in
suspension they can be plated and transfected to form
attached clones on plates. Once the clones are
isolated, they can still grow in suspension. For your
cells; first try to plate them and try to see whether
they are attaching-although loosely they may attach
enough to allow transfection- then try transfecting in
plates.

If your cell line does not allow this, then another
alternative is using an IRES-GFP construct to express
your gene/shRNA of interest. Alternatively you can
cotransfect your construct with a pGFP vector, albeit
this may bring some false positives. Once trasfected
and selected one-two weeks in the selective medium
then sort the GFP expressing cells by flow cytometry.
This would give you a polyclonal but stable cell
population.

Alternatively, some people used IRES-cell surface
marker construct to establish stable suspension cells.
In this case stable cells co-express a well
characterized heterogenous cell surface marker which
allows you to use a specific antibody against this
surface antigen coupled to magnetic beads. A couple of
rounds of sepearation over a magnetic platform
provides a stable cell population as well. If desired,
this approach can be combined with drug selection to
ensure the inhibition of the growth of any "spurious"
cells.

For details of these procedures please refer to some
landmark chapters in Methods in Enzymology.

I hope this may be of any help
Good luck,

Ozan  


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